Protective immune response against newly emerging goose tembusu virus infection induced by immunization with a recombinant envelope protein.

UNLABELLED: Flavivirus envelope protein locates at the outermost surface of viral particle and mediates virus entry and fusion infection, and plays an important role in eliciting neutralizing antibody. In this study, goose tembusu virus (GTV) E protein was expressed and purified from Escherichia coli. The immunological changes and protection efficiency of this protein serving as recombinant subunit vaccine were then tested. In ducks, recombinant E protein markedly elicited specific neutralizing antibody, stimulated the secretion of IL-2, TNF-α and IFN-γ cytokines and promoted the proliferation of lymphocytes. Additionally, population of soluble CD4 and soluble CD8 molecules in sera significantly increased in response to this antigen. Moreover, virus challenge revealed that recombinant E protein contributed to protection against GTV challenge. Taken together, the data indicate that recombinant GTV E protein could induce significant humoral and cell-mediated responses and that recombinant E protein can serve as a potential subunit vaccine candidate against GTV. SIGNIFICANCE AND IMPACT OF THE STUDY: The sudden outbreak and quick spread of newly emerging goose tembusu virus (GTV) have resulted in serious economic loss. There is no effective commercial vaccine or reasonably available control measure so far. In this study, GTV E protein was expressed and purified from Escherichia coli, and it was found that recombinant E protein could induce significant humoral and cell-mediated responses. It indicated that recombinant E protein can serve as a potential subunit vaccine candidate against GTV.

Transforming growth factor-beta: recent advances on its role in immune tolerance.

Transforming growth factor (TGF-ß1) is a pleiotropic cytokine, secreted by immune and nonhematopoietic cells. TGF-ß is involved in many different critical processes, such as embryonal development, cellular maturation and differentiation, wound healing, and immune regulation. It maintains immune homeostasis by acting as a potent immune suppressor through inhibition of proliferation, differentiation, activation, and effector function of immune cells. Paradoxically, depending on the context, it displays proinflammatory properties by being a potent chemoattractant for neutrophils and promoting inflammation. In addition, it does not only induce differentiation into the anti-inflammatory Treg cells, but also into the proinflammatory Th17 and Th9 cells and inhibits Th22 differentiation. TGF-ß has been demonstrated to be involved in multiple pathologies. In infections, it protects against collateral damages caused by the immune system, but it also promotes immune evasion and chronic infections. In autoimmune diseases, a TGF-ß dysfunction leads to the loss of tolerance to self-antigens. In cancer, TGF-ß is a potent inhibitor of cell proliferation and acts as a tumor suppressor at the beginning of tumorogenesis. However, once the cells become resistant to TGF-ß, it mainly supports tumor growth and metastasis by promoting immune evasion and angiogenesis. In asthma, it is assumed to promote allergen tolerance, but plays a detrimental role in irreversible remodeling of the airways. Despite the high numbers of TGF-ß-targeted pathways, it is a promising drug target for treatment of autoimmunity, cancer, fibrosis, if cell specificity can be achieved.This review summarizes the progresses that have been accomplished on the understanding of TGF-ß's signaling in the immune homeostasis and its role in pathogenesis.

Chimeric receptors with 4-1BB signaling capacity provoke potent cytotoxicity against acute lymphoblastic leukemia.

To develop a therapy for drug-resistant B-lineage acute lymphoblastic leukemia (ALL), we transduced T lymphocytes with anti-CD19 chimeric receptors, consisting of an anti-CD19 single-chain variable domain (reactive with most ALL cases), the hinge and transmembrane domains of CD8alpha, and the signaling domain of CD3zeta. We compared the antileukemic activity mediated by a novel receptor ('anti-CD19-BB-zeta') containing the signaling domain of 4-1BB (CD137; a crucial molecule for T-cell antitumor activity) to that of a receptor lacking costimulatory molecules. Retroviral transduction produced efficient and durable receptor expression in human T cells. Lymphocytes expressing anti-CD19-BB-zeta receptors exerted powerful and specific cytotoxicity against ALL cells, which was superior to that of lymphocytes with receptors lacking 4-1BB. Anti-CD19-BB-zeta lymphocytes were remarkably effective in cocultures with bone marrow mesenchymal cells, and against leukemic cells from patients with drug-resistant ALL: as few as 1% anti-CD19-BB-zeta-transduced T cells eliminated most ALL cells within 5 days. These cells also expanded and produced interleukin-2 in response to ALL cells at much higher rates than those of lymphocytes expressing equivalent receptors lacking 4-1BB. We conclude that anti-CD19 chimeric receptors containing 4-1BB are a powerful new tool for T-cell therapy of B-lineage ALL and other CD19+ B-lymphoid malignancies.

Exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) renders influenza virus-specific CD8+ T cells hyporesponsive to antigen.

While considerable evidence indicates that exposure to the pollutant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) impairs T cell function, the precise mechanism underlying this effect is not well understood. Furthermore, relatively little is known about the effects of TCDD on the fate of activated, antigen-specific T cells in vivo. In the present study, we took advantage of major histocompatibility complex (MHC) class I-restricted tetramers and clonotypic anti-T cell receptor (TCR) antibodies to follow the fate of influenza virus-specific CD8+ T cells in mice treated with TCDD. Exposure to TCDD suppressed the clonal expansion of influenza virus-specific CD8+ T cells, resulting in a three- to five-fold reduction in the number of cytotoxic T lymphocytes (CTL) in the lymph node, as compared to vehicle-treated mice. Studies to address possible mechanisms for the diminished CTL response failed to show evidence for increased apoptosis in virus-specific CD8+ T cells from TCDD-exposed mice. However, treatment with TCDD reduced the number of proliferating virus-specific CD8+ T cells by as much as 70% on day 7 post infection. Moreover, ex vivo restimulation of lymph node cells with influenza virus nucleoprotein (NP366-374) peptide and exogenous interleukin-2 (IL-2) only partially restored the proliferation of influenza virus-specific CD8+ T cells from TCDD-exposed mice and failed to stimulate interferon-gamma (IFNgamma) production by these cells. The observation that neither proliferation nor IFNgamma production by CD8+ T cells could be completely restored, even when cells were provided with optimal stimulation, suggests that exposure to TCDD drives antigen-specific CD8+ T cells into a state of unresponsiveness similar to anergy.

Soluble HLA class I molecules/CD8 ligation trigger apoptosis of CD8+ cells by Fas/Fas-ligand interaction.

The human major histocompatibility complex (HLA) encodes two sets of HLA class I molecules, which have been termed class Ia (or classical) and class Ib (or nonclassical) molecules. The class Ia molecules include the gene products of HLA-A, HLA-B, and HLA-C loci and are characterized by broad tissue expression and by a high degree of polymorphism. The class Ib molecules include the gene products of HLA-E, HLA-F, and HLA-G loci and are characterized by a restricted tissue distribution and by limited polymorphism. Besides being expressed on nucleated cells, classical and nonclassical HLA class I molecules are present in serum in soluble form (sHLA-I). The serum level of sHLA-I molecules is significantly increased in a variety of physiological and pathological conditions such as pregnancy, acute rejection episodes following organ allografts, acute graft-versus-host-disease (GVHD) following bone marrow transplantation, autoimmune diseases, viral infections, and malignant melanoma. Because of the statistically significant association with clinical parameters, the level of sHLA-I antigens has been suggested to represent a useful marker to predict the evolution of viral infections and to monitor the clinical course of allografts. Moreover, elevated levels of functional sHLA-I and soluble Fas-ligand molecules have been detected by our group in blood components and might play a role in the immunomodulatory effect of autologous and allogeneic transfusions. Several lines of evidence suggest that sHLA-I molecules are immunologically functional and may play an immunoregulatory role. In fact, they have been shown to elicit antibodies in both allogeneic and xenogeneic combinations, to inhibit the activity of alloreactive cytotoxic T lymphocytes (CTL), and to induce apoptosis in alloreactive and virus-specific CTL, in activated autologous and allogeneic CD8+ T cells, and in CD8+ NK cells. There is general agreement about the mechanism underlying the inhibition of CTL activity by sHLA antigens. This inhibition appears to be mediated by interactions of sHLA-I antigens a1 and a2 domains with T cell receptor (TCR). By contrast, there is conflicting information about the mechanism underlying induction of apoptosis of activated T cells by sHLA-I antigens. Several authors reported that sHLA-I molecules induced apoptosis of alloreactive CD8+ cytotoxic T lymphocytes through interaction with their TCR. However, our own data and those other groups indicate that classical and nonclassical sHLA-I molecules trigger Fas/Fas-ligand mediated apoptosis of phytohemoagglutinin (PHA)-activated and virus-specific CD8+ T lymphocytes as well as of CD8+ NK cells by interacting with CD8 coreceptor. Recently, we performed a series of experiments in our laboratory to clarify the intracellular mechanism(s) leading to Fas-ligand upregulation and secretion. These unpublished data indicate that sHLA-I/CD8 ligation elicits the phosphorylation of p56lck protein thyrosin kinase (PTK) associated with CD8 cytoplasmic domain in the absence of any other TCR-derived signal, the activation of syk-like ZAP-70 PTK and protein kinase C, and extracellular calcium influx. Then, activation and nuclear translocation of NF-kB and NF-AT occurs, leading to Fas-ligand mRNA transcription and soluble Fas-ligand secretion, which delivers the death signal. Interestingly, soluble Fas-ligand secretion and CD8+ cell apoptosis, but not CD8+ cell cytolitic activity, are completely inhibited by Cyclosporin A, which specifically blocks the activation of the calcineurin/calmodulin pathway. Taken together, these data suggest that sHLA-I molecules are involved in a signal-transduction pathway leading to Fas-ligand expression, soluble Fas-ligand secretion, and CD8+ cells apoptosis.

Antagonism of cytotoxic T-lymphocyte activation by soluble CD8.

The CD8 co-receptor is important in the differentiation and selection of class I MHC-restricted T cells during thymic development, and in the activation of mature T lymphocytes in response to antigen. Here we show that soluble CD8alphaalpha receptor, despite an extremely low affinity for MHC, inhibits activation of cytotoxic lymphocytes by obstructing CD3 zeta-chain phosphorylation. We propose a model for this effect that involves interference of productive receptor multimerization at the T-cell surface. These results provide new insights into the mechanism of T-cell activation and evidence that CD8 function is exquisitely sensitive to disruption, an effect that might be exploited by molecular therapeutics.

A structure-based approach to designing synthetic CD8alpha peptides that can inhibit cytotoxic T-lymphocyte responses.

CD8 molecules function as co-receptors on cytotoxic T lymphocytes (CTLs), interacting with a nonpolymorphic region of the major histocompatibility complex (MHC) class I a3 domain on antigen-presenting cells. Analogues were designed from a structural model of the mouse CD8a molecule to identify surfaces involved in CD8 function. Peptides were screened for in vitro biological activity on alloreactive CTLs, and analogue SC4 (p54-59) was found to be inhibitory during both the generation and effector stages. SC4 was also able to significantly prolong skin allograft survival across a MHC class I barrier. Thus, such CD8 analogues may have therapeutic potential as immunoregulators of CTL immune responses.

Regulatory role of CD8 in major histocompatibility complex-unrestricted tumoricidal activity of mouse T cells activated with anti-CD3 monoclonal antibody.

Antigen-nonspecific CD8+ cytotoxic T cells induced with anti-CD3 monoclonal antibody (mAb) are able to kill tumor cells in a major histocompatibility complex (MHC)-unrestricted fashion. However, the role of CD8 in the MHC-independent tumoricidal activity of anti-CD3-activated killer T (AK-T) cells has not been investigated. Here we show that anti-CD8 alpha mAb inhibits, in a dose-dependent fashion, lysis of P815 and YAC-1 tumor cells by mouse AK-T cells. The inhibition of MHC-unrestricted cytotoxicity by anti-CD8 alpha mAb cannot be attributed to interference with an adhesion-like function of CD8 towards class I MHC molecules on the target cells because anti-CD8 alpha mAb (i) had equal inhibitory effects on the cytolysis of tumor target cells regardless of their relative level of class I MHC molecule expression and (ii) did not interfere with the formation of conjugates between AK-T cells and class I MHC-bearing P815 tumor cells. However, anti-CD8 alpha mAb abrogated AK-T cell granule exocytosis in the presence of P815 tumor cells, indicating a regulatory role for CD8 in the signal transduction events which result in lysis of the tumor target cells. Immunoblot analysis of the post-nuclear fraction of lysates from AK-T cells exposed to P815 tumor cells in the presence of anti-CD8 alpha mAb revealed reduced phosphorylation of tyrosine residues on a protein with an Mr of approximately 62 kDa. Taken together, these data suggest that CD8 is able to affect the tumoricidal activity of MHC-unrestricted AK-T cells independent of class I MHC molecules on the target cell.

Influence on CD8 of TCR/CD3-generated signals in CTL clones and CTL precursor cells.

Alloreactive CTL clones and naive CTL precursor cells (CTLp) from TCR-transgenic mice were analyzed for their response in total and in TCR-associated kinase activation upon stimulation with the relevant class I allo-APCs. The responses were found to be stronger and more sustained for the CTL clone and CTLp expressing a TCR previously characterized as CD8 coreceptor independent than for the CTL clone and CTLp expressing a TCR characterized as CD8 dependent. Unexpectedly, it was found that also in response to CD3 engagement, total and TCR-associated kinase activation were stronger and more sustained in the CTL clone and CTLp expressing the CD8-independent TCR. In both types of CTL clones, p56(lck) was found associated with the TCR complex, and CD3 components were found associated with CD8 before CD3 engagement. Upon CD3 engagement, ZAP-70 was also found associated with the TCR complex and the kinase activity (p56(lck)) associated with CD8 increased. This increase was more pronounced for the CD8-independent than for the CD8-dependent clone. An increased association of CD3zeta with CD8 was also detected after CD3 engagement for each clone. These data indicate that signals resulting from exclusive CD3 engagement can influence CD8 molecular associations and activate CD8-bound p56(lck). They further suggest that clonal differences exist that influence the efficiency of signaling upon binding of the same CD3 ligand. The observation that this property was shared between independently derived CTL clone and CTLp expressing the same TCR suggests that it may be acquired during repertoire selection.

The CD8 beta polypeptide is required for the recognition of an altered peptide ligand as an agonist.

T cell activation is triggered by the specific recognition of cognate peptides presented by MHC molecules. Altered peptide ligands are analogs of cognate peptides which have a high affinity for MHC molecules. Some of them induce complete T cell responses, i.e. they act as agonists, whereas others behave as partial agonists or even as antagonists. Here, we analyzed both early (intracellular Ca2+ mobilization), and late (interleukin-2 production) signal transduction events induced by a cognate peptide or a corresponding altered peptide ligand using T cell hybridomas expressing or not the CD8 alpha and beta chains. With a video imaging system, we showed that the intracellular Ca2+ response to an altered peptide ligand induces the appearance of a characteristic sustained intracellular Ca2+ concentration gradient which can be detected shortly after T cell interaction with antigen-presenting cells. We also provide evidence that the same altered peptide ligand can be seen either as an agonist or a partial agonist, depending on the presence of CD8beta in the CD8 co-receptor dimers expressed at the T cell surface.

CD43 expression in a B cell lymphoma, WEHI 231, reduces susceptibility to G1 arrest and extends survival in culture upon serum depletion.

CD43 is a major surface sialoprotein on hemopoietic cells, whose extracellular domain is heavily O-glycosylated. The functional role of CD43 in the hemopoietic system is not fully understood; however, it has been suggested that CD43 may have a role in cell-cell repulsion and in modifying T cell proliferation and activation. CD43 is expressed in immature B cells in the bone marrow, but not by peripheral B cells, except for B-1 B cells and plasma cells. To analyze the biological effect of CD43 in B-lineage cells, we transfected mouse CD43 cDNA into a CD43- B cell lymphoma, WEHI 231, and the growth and survival in culture were compared to those of a parental cell line, human CD8 transfectants, and CD43- revertants established from CD43+ clones. We observed that CD43 expression supported cell growth in culture upon serum reduction, whereas growth of CD43- cell lines was barely detected under this condition. CD43- cell lines accumulated in G1 phase of the cell cycle, and the numbers of viable cells were greatly reduced during culture upon serum depletion, whereas expression of CD43 reduced the susceptibility to G1 arrest and temporarily retarded the apoptotic process, which, in turn, resulted in an increase and maintenance of the number of viable cells in culture. The results suggest that CD43 may have some role in the survival and expansion of B-lineage cells. The biological effect of CD43 was initiated without stimulation by cross-linking and was significantly impaired by replacement of the extracellular domain by the human CD8 extracellular domain. The basis of these regulatory processes is discussed.

Identification of a human CD4-CDR3-like surface involved in CD4+ T cell function.

The CD4 molecule is expressed on the surface of helper T cells. This molecule contains four tandem external immunoglobulin-like domains (D1-D4), a transmembrane domain, and a cytoplasmic tail. Through the use of molecular modeling techniques, peptide analogs of the CDR3-like region of the human CD4 molecule, analog hPGP, a cyclized peptide 13 amino acids long, was synthesized and tested for its ability to inhibit proliferation in human mixed lymphocyte reactions. A conservative amino acid substitution was made at position 5 (D --> N) to increase its activity and designated hPGP(N). A series of alanine substitution peptides were synthesized based on the sequence of hPGP(N) to determine the importance of each residue to the peptide's function. The substitutions of amino acids in positions 3, 7, and 8 had essentially no effect on the inhibitory activity of hPGP(N), while substitutions of amino acids in positions 4 and 6 increased its inhibitory effect. Alanine substitutions of amino acids in positions 2, 5, and 9 dramatically decreased the inhibitory effect of analog hPGP(N). Molecular modeling of the native CD4-CDR3-like domain suggested that the residues corresponding to positions 2, 5, and 9 of the peptide formed a contiguous surface representing the active site.

CD8+ T cells suppress human immunodeficiency virus replication by inhibiting viral transcription.

CD8+ cells from human immunodeficiency virus (HIV)-infected individuals suppress HIV replication in cultured CD4+ cells by a noncytolytic mechanism that involves a secreted CD8(+)-cell antiviral factor (CAF). The results of this study suggest that CD8+ cells, as well as CAF, arrest HIV replication at the level of viral transcription. Culturing naturally infected CD4+ cells actively producing HIV with autologous CD8+ cells or a 50% dilution of culture fluids from these cells results in a > 80% reduction in the number of cells expressing HIV antigens and RNA. This effect was observed within 2 days after exposure to CD8+ cells but required 6 days in the presence of CAF-containing culture fluids to reach the same extent of HIV suppression. Northern blot analysis of CD4+ cell extracts revealed that all viral RNA species (unspliced and single and double spliced) were reduced in quantity to a similar extent. CAF-containing culture fluids also had a direct inhibitory effect on HIV long terminal repeat (LTR)-driven transcription in HIV-infected 1G5 cells carrying an LTR-luciferase construct. Suppression of basal levels of LTR-driven transcription was not detected. Thus, the results suggest that the noncytolytic CD8+ cell antiviral activity observed in HIV infection exerts its effects, at least in part, by specifically interrupting HIV transcription. These findings could help in developing therapies for HIV infection.

CD8 is critically involved in lymphocyte activation by a T. brucei brucei-released molecule.

T. brucei brucei released a lymphocyte triggering factor (TLTF), which triggered purified CD8+, but not CD4+, T cells to interferon gamma (IFN-gamma) mRNA expression and secretion and to [3H]thymidine incorporation. TLTF also induced mRNA for transforming growth factor beta, but not for interleukin-4. The action of this TLTF on mononuclear cell (MNC) cultures was blocked by anti-CD8 antibodies and by soluble CD8. MNCs from a mutant mouse strain lacking CD8 expression were not triggered by TLTF. IFN-gamma provides a growth stimulus for T. brucei brucei, and infected CD8- mice had much lower parasitemia and survived longer than CD8+ mice. The host-parasite interaction in experimental African trypanosomiasis thus involves parasite release of TLTF, which by binding to CD8 triggers CD8+ cells to produce the parasite growth-promoting cytokine IFN-gamma.