The Jβ segment of the T cell receptor contributes to the Vβ-specific T cell expansion caused by staphylococcal enterotoxin B and Urtica dioica superantigens

We have used a new polymerase chain reaction-based technique to analyze at the clonal level the CDR3 diversity and the Jβ usage associated with the Vβ-dependent T cell receptor (TCR) recognition of two superantigens: the staphylococcal enterotoxin B and the Urtica dioica agglutinin. Our results show that a subset of Jβ elements is preferentially expanded in a given Vβ family, independently of the nature of the superantigen. By contrast, the CDR3 loop does not contribute significantly to the T cell expansion induced by the superantigens. We conclude that the Jβ segment of the TCR β chain, but not the CDR3 region, participates in superantigen binding, presumably by influencing the quaternary structure of the TCR β chain.     

Influenza A virus hemagglutinin is a B cell-superstimulatory lectin

Influenza A viruses display T cell-independent polyclonal B cell-activating properties which are mediated by the B cell-superstimulatory envelope glycoprotein hemagglutinin (HA). In this report, the receptor-binding requirements for B cell activation by influenza viruses were expected. Neuraminidase treatment of resting mature B cells from BALB/c mice abrogated late (proliferation/immunoglobulin synthesis), early (up-regulation of cell surface markers, including CD25, B220, and B7-1) and veryearly events (homotypic adhesion) in virus-responding B lymphocytes. Similarly, pretreatment of murine responder cells with different inhibitors ofN-glycosylation (tunicamycin, deoxymannojirimycin) significantly suppressed subsequent B lymphocyte activation by HA, but not control responses to lipopolysaccharide or anti-μ. Assays with chimeric HA transfectants, expressing the loop region of epitope B (amino acids 155–160) of the globular head of H2 (high B cell-stimulatory subtype) or H3 (medium-stimulatory subtype) on the protein backbone of a low-stimulatory subtype (H1) failed to alter the B cell-stimulatory activity of the virus, suggesting that the hypervariable loop region is not crucial in determining the B cell-activating properties of the protein. Collectively, our results imply that the B cell-superstimulatory function of influenza virus HA is not mediated by a direct protein/protein interaction, but via binding of HA to terminal sialic acid residues on cell surface receptor glycoproteins. These findings identify the influenza virus HA glycoprotein as the first viral lectin with lymphocyte-activating properties.     

肝癌靶向性超抗原表达载体的构建与鉴定

目的：设计并构建受AFP基因顺式作用元件调控，并经减毒处理的肝癌特异性超抗原表达载体。方法：用新设计的引物作PCR扩增截短的AFP基因启动子和增强子、linker-CD80tm和SEA（D227A）。将上述片段与逆转录病毒载体pLXSN的多克隆位点连接，构建成为AFP基因顺式作用元件调控的肝癌特异性减毒超抗原表达载体[pLXSN SEA(D227A)-Linker-CD80tm]，并用软件对其开放读框进行分析。结果：成功克隆了截短的AFP基因启动子、增强子、linker-CD80tm和SEA（D227A）。将上述序列连接到逆转录病毒载体pLXSN的多克隆位点，酶切鉴定和DNA序列分析无误。结论：AFP基因转录启动元件修饰的SEA表达载体，在基因转录水平定量、定向、特异性调控强有力的细胞和体液免疫活化剂（SEA），为下一步将其作为基因疫苗治疗肝癌奠定了基础。     

Delayed allograft rejection by T cell receptor Vβ8.1 transgenic mice peripherally tolerized to Mls-1

One commonly studied model system for peripheral tolerance is the antigen-specific unresponsiveness of T cells from mice previously inoculated with superantigens such as Mls-1^a. In this study, we used a TcR Vβ8.1 transgenic mouse model to investigate whether mice peripherally tolerized to Mls-1^a exhibit delayed skin allograft rejection. We report dramatic prolongation of skin allograft survival in Vβ8.1 transgenic but not in non-transgenic mice tolerized to Mls-1^a. Peripherally induced unresponsiveness to Mls-1^a can, therefore, be considered true tolerance.     

Superantigens initiate cognate CD4+ T cell/ B cell interactions leading to early activation and proliferation of B cells

Dimerization or even multimerization of various receptors is commonly required for signal transduction. We report here that clustering of major histocompatibility complex class II molecules in human B cells by biotinylated staphylococcal enterotoxin A (SEA) cross-linked with avidin induces an increase in the level of intracellular calcium. This response was abolished by prior treatment with protein tyrosine kinase (PTK) inhibitors, suggesting that SEA-triggered calcium mobilization in B cells is probably dependent on the activation of PTK. The implication of PTK in SEA-induced early B cell activation was then confirmed by demonstrating that cross-linked SEA induces a significant increase in the level of tyrosine phosphorylation in B cells. The requirement of biotinavidin cross-linking in SEA-induced calcium mobilization in B cells can be fulfilled by the addition CD4⁺ T cells, suggesting a role for CD4 molecules. Using the murine CD4^? T cell hybridoma 3DT, or its derivative 11B3 transfected with human CD4 that both express SEA-specific TCR, we confirmed the CD4 requirement for B cell calcium mobilization and that both specific TCR and CD4 molecules are required in early events of B cell activation induced by SEA. The role of CD4 in SEA-induced B cell proliferation was then investigated. SEA-stimulated B cells proliferated in the presence of CD4⁺ T cells, whereas no response was observed in the presence of CD8⁺ T cells. The addition of clone 11B3 CD4⁺ T cells failed to fulfill the requirement of CD4⁺ T cells in SEA-induced B cell proliferation, indicating the possible involvement of other CD4⁺ T cell surface molecules in this response. This issue is currently under investigation.     

T cells made deficient in interleukin-2 production by exposure to staphylococcal enterotoxin B in vivo are primed for interferon-γ and interleukin-10 secretion

The superantigen staphylococcal enterotoxin B (SEB) induces a defect in interleukin (IL)-2 production by T cells expressing specific T cell receptor Vβ domains. The present study was undertaken to determine the capacity of T cells, made deficient in IL-2 production by exposure to SEB in vivo, to secrete interferon (IFN)-γ and IL-10 and to induce pathology upon SEB rechallenge. For this purpose, BALB/c mice received two intraperitoneal injections of 100 μg SEB with a 48-h interval. First, we compared peak serum levels of IL-2, IFN-γ and IL-10 after SEB rechallenge with those measured after a single SEB injection in control mice. The expected defect in IL-2 production in SEB-pretreated mice was associated with a major increase in IL-10 and IFN-γ levels which were about fivefold higher than in controls. Experiments in mice depleted of CD4⁺ or CD8⁺ cells as well as studies in which purified T cell populations were rechallenged with SEB in vitro indicated that both CD4⁺ and CD8⁺ cells from SEB-pretreated mice were primed for IL-10 and IFN-γ production. Furthermore, SEB-pretreated mice were sensitized to the toxic effects of the superantigen as indicated by a 30-70% lethality rate (vs. 0% in naive mice) within 48 h after SEB rechallenge. IFN-γ was involved in the lethal syndrome as it could be prevented by injection of neutralizing anti-IFN-γ monoclonal antibody. We conclude that SEB-reactive T cells made deficient for the production of IL-2 by exposure to SEB in vivo are primed for IFN-γ and IL-10 production, and that IFN-γ up-regulation is involved in the shock syndrome occurring upon SEB rechallenge.     

A Vβ.2-specific superantigen from exogenous mouse mammary tumor virus carried by FM mice

A number of endogenous mouse mammary tumor virus (MMTV) proviruses encode superantigen that have the ability to stimulate T cells with a certain T cell receptor (TCR) β-chain variable region (Vβ) and to mediate the Vβ-specific clonal deletion. The tumorigenic milk-borne MMTV carried by C3H and GR mice also have superantigenic properties in vivo. In the present study we identified and characterized a novel Vβ8.2-specific superantigen of exogenous MMTV carried by FM mice. The open reading frame (ORF) in the 3′ long terminal repeat of the MMTV was cloned by polymerase chain reaction with primers corresponding to conserved regions spanning the ORF coding region. Sequence analysis of the ORF revealed that there is no sequence identical to those in other known MMTV in the carboxy terminus implicated in TCR Vβ recognition. Subcutaneous injection of the virus into adult BALB/c mice induced an approximately three- to fourfold enlargement of draining lymph nodes and a substantial increase of Vβ8.2⁺ CD4⁺ T cells in the lymph nodes within 6 days. The exposure of newborn BALB/c mice to the virus by foster nursing resulted in a marked deletion of Vβ8.2⁺ cells both in CD4⁺ and CD8⁺ T cells. Thus, a novel milk-borne MMTV in FM mice expresses strong superantigenic properties capable of stimulating Vβ8.2⁺ T cells. Vβ8.2⁺ T cells have been demonstrated to be frequently involved in recognition of conventional antigens and responsible for autoimmune diseases such as experimental allergic encephalomyelitis. Therefore, the MMTV (FM) may provide a new mouse model system for inducing immunodeficiency or autoimmune disease by retroviral infection.     

Roles of Toll-like receptor 2 (TLR2) and superantigens on adaptive immune responses during CNS staphylococcal infection

Staphylococcus aureus is a common etiologic agent of brain abscesses and possesses numerous virulence factors that manipulate host immunity. One example is superantigens (SAG) that clonally expand T cell subsets bearing specific Vβ receptors. Toll-like receptor 2 (TLR2) is one receptor implicated in S. aureus recognition. However, the interplay between TLR2, SAG, and adaptive immunity during brain abscess formation has not yet been investigated and could reveal novel insights into host-pathogen interactions for regulating protective immunity. A comprehensive analysis of abscess-associated T cell populations in TLR2 KO and WT mice was performed following infection with a S. aureus clinical isolate. Both natural killer T (NKT) and γδ T cell infiltrates were increased in brain abscesses of TLR2 KO mice and produced more IL-17 and IFN-γ compared to WT populations, which could have resulted from elevated bacterial burdens observed in these animals. Analysis of SAG-reactive T cells revealed a predominant Vβ_8.1,8.2 infiltrate reactive with staphylococcal enterotoxin B (SEB), whereas SEA-reactive Vβ₁₁ T cells were less numerous. Brain abscesses of TLR2 KO mice had fewer Vβ_8.1,8.2 and Vβ₁₁ T cells and produced less TNF-α and IFN-γ compared to WT animals. Treatment of primary microglia with purified SEB augmented TNF-α production in response to the TLR2 ligand Pam3Cys, which may serve to amplify proinflammatory cascades during CNS S. aureus infection. Collectively, these studies demonstrate that TLR2 impacts adaptive immunity to S. aureus infection and modulates SAG responses.