Photophysical analysis of class I major histocompatibility complex protein assembly using a xanthene-derivatized beta2-microglobulin. (9/5440)

Spectral changes and a sixfold increase in the emission intensity were observed in the fluorescence of a single xanthene probe (Texas red) attached to beta2m-microglobulin (beta2m) upon assembly of beta2m into a ternary complex with mouse H-2Kd heavy chain and influenza nuclear protein peptide. Dissociation of the labeled beta2m from the ternary complex restored the probe's fluorescence and absorption spectra and reduced the emission intensity. Thus changes in xanthene probe fluorescence upon association/dissociation of the labeled beta2m molecule with/from the ternary complex provide a simple and convenient method for studying the assembly/dissociation mechanism of the class I major histocompatibility complex (MHC-I) encoded molecule. The photophysical changes in the probe can be accounted for by the oligomerization of free labeled beta2m molecules. The fluorescence at 610 nm is due to beta2m dimers, where the probes are significantly separated spatially so that their emission and excitation properties are close to those of xanthene monomers. Fluorescence around 630 nm is due to beta2m oligomers where xanthene probes interact. Minima in the steady-state excitation (550 nm) and emission (630 nm) anisotropy spectra correlate with the maxima of the high-order oligomer excitation and emission spectra, showing that their fluorescence is more depolarized. These photophysical features are explained by splitting of the first singlet excited state of interacting xanthene probes that can be modeled by exciton theory.  (+info)

Cytotoxicity is mandatory for CD8(+) T cell-mediated contact hypersensitivity. (10/5440)

Contact hypersensitivity (CHS) is a T cell-mediated skin inflammation induced by epicutaneous exposure to haptens in sensitized individuals. We have previously reported that CHS to dinitrofluorobenzene in mice is mediated by major histocompatibility complex (MHC) class I-restricted CD8(+) T cells. In this study, we show that CD8(+) T cells mediate the skin inflammation through their cytotoxic activity. The contribution of specific cytotoxic T lymphocytes (CTLs) to the CHS reaction was examined both in vivo and in vitro, using mice deficient in perforin and/or Fas/Fas ligand (FasL) pathways involved in cytotoxicity. Mice double deficient in perforin and FasL were able to develop hapten-specific CD8(+) T cells in the lymphoid organs but did not show CHS reaction. However, they did not generate hapten-specific CTLs, demonstrating that the CHS reaction is dependent on cytotoxic activity. In contrast, Fas-deficient lpr mice, FasL-deficient gld mice, and perforin-deficient mice developed a normal CHS reaction and were able to generate hapten-specific CTLs, suggesting that CHS requires either the Fas/FasL or the perforin pathway. This was confirmed by in vitro studies showing that the hapten-specific CTL activity was exclusively mediated by MHC class I-restricted CD8(+) T cells which could use either the perforin or the Fas/FasL pathway for their lytic activity. Thus, cytotoxic CD8(+) T cells, commonly implicated in the host defence against tumors and viral infections, could also mediate harmful delayed-type hypersensitivity reactions.  (+info)

Maturation, activation, and protection of dendritic cells induced by double-stranded RNA. (11/5440)

The initiation of an immune response is critically dependent on the activation of dendritic cells (DCs). This process is triggered by surface receptors specific for inflammatory cytokines or for conserved patterns characteristic of infectious agents. Here we show that human DCs are activated by influenza virus infection and by double-stranded (ds)RNA. This activation results not only in increased antigen presentation and T cell stimulatory capacity, but also in resistance to the cytopathic effect of the virus, mediated by the production of type I interferon, and upregulation of MxA. Because dsRNA stimulates both maturation and resistance, DCs can serve as altruistic antigen-presenting cells capable of sustaining viral antigen production while acquiring the capacity to trigger naive T cells and drive polarized T helper cell type 1 responses.  (+info)

Characterization of CD4+ CD8alphaalpha+ and CD4-CD8alphaalpha+ intestinal intraepithelial lymphocytes in rats. (12/5440)

Intestinal intraepithelial lymphocytes (i-IEL) of aged rats comprise CD4+CD8alphaalpha+ and CD4-CD8alphaalpha+ T cells expressing TCR alphabeta. In the present study, we compared characteristics between CD4+CD8alphaalpha+ and CD4-CD8alphaalpha+ i-IEL, which were purified by a cell sorter from the i-IEL of 6-month-old Lewis rats. Most of the CD4+CD8alphaalpha+ i-IEL were of the CD44(hlgh) phenotype, while CD4-CD8alphabeta+ i-IEL were CD44(low). Vbeta usage in the CD4-CD8alphaalpha+ i-IEL was much diversified, while CD4+CD8alphaalpha+ i-IEL showed a skewed Vbeta repertoire. The CD4+CD8alphaalpha+ i-IEL but not the CD4-CD8alphaalpha+ i-IEL proliferated in response to syngeneic spleen cells, which was partially inhibited by addition of anti-MHC class I mAb. The CD4+CD8alphaalpha+ i-IEL produced IFN-gamma and IL-2 but no IL-4 or transforming growth factor (TGF)-beta in response to syngeneic spleen cells, while CD4-CD8alphaalpha+ i-IEL produced abundant levels of TGF-beta but no IL-2, IFN-gamma or IL-4. CD4+CD8alphaalpha+ i-IEL proliferated in response to exogenous IL-2 but not to IL-15, while CD4-CD8alphaalpha+ i-IEL could respond to IL-15 as well as IL-2. These results suggest that a significant fraction of CD4+CD8alphaalpha+ i-IEL belongs to Th1-type T cells capable of responding to self-MHC class I, while CD4-CD8alphaalpha+ i-IEL are a unique population with a diversified Vbeta repertoire that respond to IL-15 in rats.  (+info)

The leukocyte Ig-like receptor (LIR)-1 for the cytomegalovirus UL18 protein displays a broad specificity for different HLA class I alleles: analysis of LIR-1 + NK cell clones. (13/5440)

Leukocyte Ig-like receptor (LIR)-1 is a member of the Ig superfamily which has been shown to bind the human cytomegalovirus MHC class I homologue UL-18 protein. In this study, we have analyzed the expression and function of LIR-1 in human NK cells. We show that LIR-1 is expressed by a subset of NK cells variable in size among different donors. When compared to the known HLA class I-specific NK receptors, the expression of LIR-1 was found to be partially overlapped with that of CD94-NKG2A or with that of killer inhibitory receptors (KIR) belonging to the Ig superfamily. The use of the soluble form of UL-18 molecule revealed, in double fluorescence analysis, a selective binding to LIR-1 + cells while no correlation was observed between expression of either KIR or CD94-NKG2A molecules and ability to bind UL18. We further determined whether LIR-1 could also function as receptor for HLA class I molecules. To this end, we assessed the capability of LIR-1 + NK cell clones of lysing HLA class I- target cells transfected with different class I alleles, including HLA-A, -B, -C and -G alleles. Data revealed that LIR-1 functions as a broad HLA class I-specific inhibitory receptor recognizing different alleles coded for by different HLA loci.  (+info)

Crossreactive recognition of viral, self, and bacterial peptide ligands by human class I-restricted cytotoxic T lymphocyte clonotypes: implications for molecular mimicry in autoimmune disease. (14/5440)

The immunodominant, CD8(+) cytotoxic T lymphocyte (CTL) response to the HLA-B8-restricted peptide, RAKFKQLL, located in the Epstein-Barr virus immediate-early antigen, BZLF1, is characterized by a diverse T cell receptor (TCR) repertoire. Here, we show that this diversity can be partitioned on the basis of crossreactive cytotoxicity patterns involving the recognition of a self peptide-RSKFRQIV-located in a serine/threonine kinase and a bacterial peptide-RRKYKQII-located in Staphylococcus aureus replication initiation protein. Thus CTL clones that recognized the viral, self, and bacterial peptides expressed a highly restricted alphabeta TCR phenotype. The CTL clones that recognized viral and self peptides were more oligoclonal, whereas clones that strictly recognized the viral peptide displayed a diverse TCR profile. Interestingly, the self and bacterial peptides equally were substantially less effective than the cognate viral peptide in sensitizing target cell lysis, and also resulted only in a weak reactivation of memory CTLs in limiting dilution assays, whereas the cognate peptide was highly immunogenic. The described crossreactions show that human antiviral, CD8(+) CTL responses can be shaped by peptide ligands derived from autoantigens and environmental bacterial antigens, thereby providing a firm structural basis for molecular mimicry involving class I-restricted CTLs in the pathogenesis of autoimmune disease.  (+info)

Interleukin-2 gene transfer into human transitional cell carcinoma of the urinary bladder. (15/5440)

Transitional cell carcinoma of the bladder is one of the human cancers most responsive to immunotherapy, and local interleukin-2 (IL-2) production appears to be an important requirement for immunotherapy to be effective. In this study, we engineered two human bladder cancer cell lines (RT112 and EJ) to constitutively release human IL-2 by retroviral vector-mediated gene transfer. Following infection and selection, stable and consistent production of biologically active IL-2 was demonstrated at both the mRNA and the protein level. Morphology, in vitro growth rate and proliferation, as well as other cytokine gene mRNA or membrane adhesion receptor expression, were not altered in IL-2 transduced cells as compared to their parental or control vector-infected counterparts. Moreover, IL-2 engineered cells lost their tumorigenicity into nu/nu mice and the mechanism of rejection appeared to involve multiple host effector cell populations, among which a prominent role was played by neutrophils and radiosensitive cells. These findings may offer support to the development of an IL-2-based gene therapy approach to human bladder cancer.  (+info)

T-cell receptor transgenic analysis of tumor-specific CD8 and CD4 responses in the eradication of solid tumors. (16/5440)

The role of tumor-specific CD8 and CD4 lymphocytes in rejecting solid tumors has been difficult to determine because of the lack of models in which tumor antigen, specific CD8 cells, and specific CD4 cells can be monitored and controlled. To investigate the minimal components required for the induction and maintenance of CTL activity sufficient to reject a solid tumor in vivo, we transfected the influenza hemagglutinin (HA) gene into a nonimmunogenic class I+/class II- murine malignant mesothelioma (MM) tumor line to generate an endogenous tumor antigen and used TCR transgenic mice with class I- or class II-restricted specificities for HA as sources of naive, tumor-specific T cells. The data show that the presence of a strong tumor antigen is not in itself sufficient to induce an effective CTL response, nor does the presence of a high frequency of precursor cells guarantee tumor rejection. We also show that tumor-specific CD4 cells, when CTL numbers are suboptimal, greatly enhance the eradication of tumor, confirming the importance of antigen-presenting cell presentation of tumor antigens to class II-restricted cells. These data confirm that T-cell receptor transgenic cells, combined with nominal tumor antigen transfection, represent powerful tools to analyze tumor-specific T-cell responses.  (+info)