Cell-cell interaction with APC, not IL-23, is required for naive CD4 cells to acquire pathogenicity during Th17 lineage commitment

Subpopulations of pathogenic or nonpathogenic Th17 cells were reported to develop when presensitized CD4 cells were activated with their target Ag during polarization by either IL-23 or IL-6 and TGF-β, respectively. In this study, we generated two Th17 subpopulations by using a system in which naive CD4 cells from TCR transgenic mice specific to hen egg lysozyme (HEL) are polarized with IL-6/TGF-β and, concurrently, are activated either with HEL presented by APCs, or with anti-CD3/CD28 Abs. Only the former cells were pathogenic, inducing inflammation in eyes expressing HEL. Naive CD4 cells activated by the anti-CD3/CD28 Abs acquired pathogenicity, however, when cocultured with HEL/APC. Importantly, the naive CD4 cells did not acquire pathogenicity when cocultured with APCs stimulated with LPS or when separated from the HEL-presenting cells by a semipermeable membrane. Unlike with presensitized Th17, soluble IL-23 does not participate in pathogenicity acquisition by naive CD4 cells; no pathogenicity was induced by adding IL-23 to cultures activated with anti-CD3/CD28 Abs. Furthermore, Abs against IL-23 or IL-23R did not inhibit acquisition of pathogenicity in cultures of naive CD4 cells activated by HEL/APC. Our data thus show that, unlike presensitized CD4 cells, naive CD4 cells polarized toward Th17 phenotype acquire pathogenicity only by direct interaction with APCs presenting the Ag, with no apparent involvement of soluble IL-23. We suggest that the Th17 lymphocytes derived from naive CD4 cells participate in pathogenic and other immune processes, along with the IL-23-dependent Th17 cells.     

Coreceptor signal strength regulates positive selection but does not determine CD4/CD8 lineage choice in a physiologic in vivo model

TCR signals drive thymocyte development, but it remains controversial what impact, if any, the intensity of those signals have on T cell differentiation in the thymus. In this study, we assess the impact of CD8 coreceptor signal strength on positive selection and CD4/CD8 lineage choice using novel gene knockin mice in which the endogenous CD8alpha gene has been re-engineered to encode the stronger signaling cytoplasmic tail of CD4, with the re-engineered CD8alpha gene referred to as CD8.4. We found that stronger signaling CD8.4 coreceptors specifically improved the efficiency of CD8-dependent positive selection and quantitatively increased the number of MHC class I (MHC-I)-specific thymocytes signaled to differentiate into CD8+ T cells, even for thymocytes expressing a single, transgenic TCR. Importantly, however, stronger signaling CD8.4 coreceptors did not alter the CD8 lineage choice of any MHC-I-specific thymocytes, even MHC-I-specific thymocytes expressing the high-affinity F5 transgenic TCR. This study documents in a physiologic in vivo model that coreceptor signal strength alters TCR-signaling thresholds for positive selection and so is a major determinant of the CD4:CD8 ratio, but it does not influence CD4/CD8 lineage choice.     

CTLA-4 is not required for induction of CD8(+) T cell anergy in vivo.

Recent studies of T cell anergy induction have produced conflicting conclusions as to the role of the negative regulatory receptor, CTLA-4. Several in vivo models of tolerance have implicated the interaction of CTLA-4 and its ligands, B7.1 and B7.2, as an essential step in induction of anergy, while results from a number of other systems have indicated that signals from the TCR/CD3 complex alone are sufficient to induce T cell unresponsiveness. One explanation for this disparity is that the requirements for anergy induction depend closely on the details of the system: in vivo vs in vitro, route of stimulus administration, naive vs memory cells, CD4(+) vs CD8(+) cells, etc. To test this possibility, we established an in vivo anergy model using mice transgenic for the 2C TCR on a recombination-activating gene-2-deficient background, that either express or lack the CTLA-4 molecule. This system provides us with a very homogeneous pool of naive Ag-specific CD8(+) T cells, allowing us to control some of the conditions mentioned above. We found that T cells from CTLA-4-deficient mice were anergized by injections of soluble antigenic peptide as efficiently as were CTLA-4-expressing cells. These results indicate that CTLA-4 is not universally required for in vivo T cell anergy induction and may point to distinctions between regulation of peripheral tolerance in CD4(+) and CD8(+) T cells.     

Early growth response (EGR)-1 is required for timely cell-cycle entry and progression in hepatocytes after acute carbon tetrachloride exposure in mice

Cell-cycle induction in hepatocytes protects from prolonged tissue damage after toxic liver injury. Early growth response (Egr)-1(-/-) mice exhibit increased liver injury after carbon tetrachloride (CCl(4)) exposure and reduced TNF-α production. Because TNF-α is required for prompt cell-cycle induction after liver injury, here, we tested the hypothesis that Egr-1 is required for timely hepatocyte entry into the cell cycle after CCl(4)-induced liver injury. Acute liver injury was induced by a single injection of CCl(4). Assays were employed to assess indices of the cell cycle in liver after CCl(4) exposure. Bromodeoxyuridine incorporation peaked in wild-type mice at 48 h after CCl(4) but was reduced by 80% in Egr-1(-/-) mice. Proliferating-cell nuclear-antigen immunohistochemistry revealed blocks in cell-cycle entry and progression to DNA synthesis in Egr-1-deficient mice 48 h after CCl(4). Cyclin D, important for G0/G1 progression, was reduced at baseline and 36 h after CCl(4). Cyclin E1, required for G1/S-phase transition, was reduced in Egr-1(-/-) mice 24 and 48 h after CCl(4) exposure and was associated with reduced phosphorylation of the retinoblastoma protein. Proliferation in Egr-1(-/-) mice was delayed, rather than blocked, because indices of cell-cycle progression were restored 72 h after CCl(4) exposure. We concluded that Egr-1 was required for prompt cell-cycle entry (G0- to G1-phase) and G1/S-phase transition after toxic liver injury. These data support the hypothesis that Egr-1 provides hepatoprotection in the CCl(4)-injured liver, attributable, in part, to timely cell-cycle induction and progression.     

Age-associated thymic atrophy is not associated with a deficiency in the CD44(+)CD25(-)CD3(-)CD4(-)CD8(-) thymocyte population.

Age-associated thymic atrophy has been proposed to be due to changes in both the thymic microenvironment and in the intrinsic properties of the early T cell progenitors, the CD44(+)CD25(-)CD3(-)CD4(-)CD8(-) cells. We have purified these cells from the thymus of both old and young mice and demonstrate no age-associated defect in their ability to differentiate into their progeny in vitro when used to reconstitute fetal thymic organ cultures. We also demonstrate that in the presence of anti-IL-7, CD44(+)CD25(-)CD3(-)CD4(-)CD8(-) cells from young mice show reduced thymocyte development in fetal thymic organ cultures compared with controls. Finally we have shown that old mice treated with IL-7 show improved thymopoiesis compared with control groups. The increased thymopoiesis seen in the old animals occurs in the sequential manner which would be anticipated for an agent working directly on the early stages, including the CD44(+)CD25(-)CD3(-)CD4(-)CD8(-) cells.     

EGR-1启动子在肿瘤基因治疗中的应用

EGR-1的启动子为早期生长反应因子-1基因上游约-550-0bp的一顺式作用元件。其活性受控于一些诱导剂,如电离辐射,联合EGR-1的启动子与治疗基因（如TNF-α、自杀基因）,用辐射能在肿瘤局部从时,空调控治疗基因的表达,使其产物局限于肿瘤局部,并发挥放疗的杀肿瘤效应,而放疗与转基因产物又有协同作用。放射治疗与基因治疗的配伍为肿瘤的治疗提供了新思路。     

Induction of the early growth response gene 1 promoter by TCR agonists and partial agonists: ligand potency is related to sustained phosphorylation of extracellular signal-related kinase substrates

Responses to partial agonist TCR signals include positive selection of thymocytes, survival of naive T cells, and homeostatic proliferation. As part of an effort to understand the molecular basis of these processes, we have determined how agonist and partial agonist ligands act differently to induce a change in gene expression. We have found that the early growth response gene 1 (Egr1) promoter is activated by agonist and partial agonist ligands, but the partial agonist induces 10-fold lower promoter activity. Both agonist and partial agonist ligands require all six serum response elements in the Egr1 promoter to reach maximum induction. Although slightly fewer cells respond to the partial agonist, all of the responding cells have reduced activity compared with the cells responding to agonist. The factors binding to the serum response elements of the Egr1 promoter form a ternary complex (TC) consisting of serum response factor and either Elk-1 or serum response factor accessory protein-1a. Formation of a stable TC and inducible promoter activity are both dependent on extracellular signal-related kinase activation. Examination of TC formation over time reveals that this complex is induced well by partial agonist ligands, but it is not sustained, whereas agonist stimulation induces longer lived TCs. Therefore, the data suggest that both agonist and partial agonist ligands can induce formation of multiple TC on the Egr1 promoter, but the ability of the agonist ligand to maintain these complexes for an extended time results in the increased potency of the agonist.     

A switch in costimulation from CD28 to 4-1BB during primary versus secondary CD8 T cell response to influenza in vivo

4-1BBL(-/-) mice exhibit normal primary CD8 T cell responses to influenza virus, but show decreased CD8 T cell numbers late in the primary response as well as decreased secondary responses. In contrast, CD28(-/-) mice are defective in initial CD8 T cell expansion. Using agonistic anti-4-1BB Ab to replace the CD28 or 4-1BB signal, we examined the timing of the required signals for CD28 vs 4-1BB costimulation. A single dose of agonistic anti-4-1BB Ab added only during priming restores the secondary CD8 T cell response in CD28(-/-) mice. Once the T cell numbers in the primary response reach a minimum threshold, a full secondary response is achieved even in the absence of CD28. In contrast, anti-4-1BB added during priming fails to correct the defective secondary response in 4-1BBL(-/-) mice, whereas addition of anti-4-1BB during challenge fully restores this response. Thus, there is a switch in costimulatory requirement from CD28 to 4-1BB during primary vs recall responses. Adoptive transfer studies show that T cells primed in 4-1BBL(-/-) or wild-type mice are equally capable of re-expansion when rechallenged in wild-type mice. These studies rule out a model in which signals delivered through 4-1BB during priming program the T cells to give a full recall response and suggest that 4-1BB-4-1BBL interactions take place at later stages in the immune response. The results indicate that anti-4-1BB or 4-1BBL therapy will be most effective during the boost phase of a prime-boost vaccination strategy.     

Early growth response-1 gene (Egr-1) promoter induction by ionizing radiation in U87 malignant glioma cells in vitro.

The promoter of the early growth response gene (Egr-1) has been described to be activated by ionizing radiation, and it seems to be clear that this process involves different mitogen activated protein (MAP) kinases, dependent on the specific cell type examined. However, early steps leading to activation of the corresponding pathways and thus to overexpression of Egr-1 are not well understood. In this study, deletion mutants of the 5' upstream region of the Egr-1 gene were generated which allowed us to correlate the radiation-induction of the Egr-1 promoter in U87 glioma cells to five serum response elements. Based on the data shown, a possible role of two cAMP responsive elements for radiation-dependent promoter regulation could be ruled out. On the basis of activator/inhibitor studies applying fetal bovine serum, EGF, PD98059, anisomycin, SB203580, forskolin and wortmannin, it could be demonstrated that in U87 cells the ERK1/2 and potentially SAPK/JNK, but not the p38MAPK/SAPK2, pathway contribute to the radiation-induction of Egr-1 promoter. In addition, it was observed that irradiated cells secrete a diffusible factor into the culture media which accounts for the radiation-induced promoter upregulation. By blocking growth factor receptor activation with suramin, this effect could be completely abolished.     

Modulation of early growth response gene 1 and interleukin-8 expression by ribotoxin deoxynivalenol (vomitoxin) via ERK1/2 in human epithelial intestine 407 cells

Epithelium senses external toxic insults transmit the sentinel signals into the cells to reprogram a broad range of mucosal responses like epithelial inflammatory diseases. The purpose of this study was to test the hypothesis that ribotoxin DON evokes the epithelial sentinel signals which contributes to the pro-inflammatory cytokine interleukin-8 in human epithelial cells. Treatment with DON elevated interleukin-8 production in the human epithelial intestine 407 cells. Particularly, ribotoxin DON markedly elevated the phosphorylated extracellular signal-regulated protein kinases 1 and 2 (ERK1/2) mitogen-activated protein kinase (MAPK) which mediated DON-induced interleukin-8 (IL-8) production. DON-activated ERK1/2 also mediated the production of early growth response gene 1 (EGR-1) in the epithelial cell line and EGR-1 had the positive regulatory effect on the interleukin-8 production in the human epithelial cells. Taken all, DON-activated MAPK sentinel signals of the epithelial cells which promoted interleukin-8 production and its positive modulator EGR-1. These findings will provide insight into the possible mechanism associated with the early epithelial inflammation by ribotoxic insults.