T cell receptor Vbeta gene usage in Thai children with dengue virus infection.

T lymphocyte activation during dengue is thought to contribute to the pathogenesis of dengue hemorrhagic fever (DHF). We examined the T cell receptor Vbeta gene usage by a reverse transcriptase-polymerase chain reaction assay during infection and after recovery in 13 children with DHF and 13 children with dengue fever (DF). There was no deletion of specific Vbeta gene families. We detected significant expansions in usage of single Vbeta families in six subjects with DHF and three subjects with DF over the course of infection, but these did not show an association with clinical diagnosis, viral serotype, or HLA alleles. Differences in Vbeta gene usage between subjects with DHF and subjects with DF were of borderline significance. These data suggest that the differences in T cell activation in DHF and DF are quantitative rather than qualitative and that T cells are activated by conventional antigen(s) and not a viral superantigen.

Identification of a crucial energetic footprint on the alpha1 helix of human histocompatibility leukocyte antigen (HLA)-A2 that provides functional interactions for recognition by tax peptide/HLA-A2-specific T cell receptors.

Structural studies have shown that class I major histocompatibility complex (MHC)-restricted peptide-specific T cell receptor (TCR)-alpha/betas make multiple contacts with the alpha1 and alpha2 helices of the MHC, but it is unclear which or how many of these interactions contribute to functional binding. We have addressed this question by performing single amino acid mutagenesis of the 15 TCR contact sites on the human histocompatibility leukocyte antigen (HLA)-A2 molecule recognized by the A6 TCR specific for the Tax peptide presented by HLA-A2. The results demonstrate that mutagenesis of only three amino acids (R65, K66, and A69) that are clustered on the alpha1 helix affected T cell recognition of the Tax/HLA-A2 complex. At least one of these three mutants affected T cell recognition by every member of a large panel of Tax/HLA-A2-specific T cell lines. Biacore measurements showed that these three HLA-A2 mutations also altered A6 TCR binding kinetics, reducing binding affinity. These results show that for Tax/HLA-A2-specific TCRs, there is a location on the central portion of the alpha1 helix that provides interactions crucial to their function with the MHC molecule.

Conversion of a T cell antagonist into an agonist by repairing a defect in the TCR/peptide/MHC interface: implications for TCR signaling.

The structure of the A6 alphabetaTCR/HTLV-1 Tax-peptide/MHC I complex with proline 6 of Tax substituted with alanine (P6A), an antagonist, is nearly identical to the structure with wild-type Tax agonist. Neither the proline in the agonist nor the alanine in the antagonist is contacted by the alphabetaTCR. Here, we demonstrate that antagonist activity of P6A is associated with low affinity of the A6 alphabetaTCR for Tax-P6A/HLA-A2. We show that stepwise repair of a packing defect in the TCR/MHC interface using N-alkylated amino acids results in stepwise increases in TCR affinity and activity. Kinetic and thermodynamic measurements suggest that for some ligands the range of T cell outcomes does not correlate with either their alphabetaTCR affinity or the half-life of the alphabetaTCR/peptide/MHC complex.

Bystander target cell lysis and cytokine production by dengue virus-specific human CD4(+) cytotoxic T-lymphocyte clones.

Dengue hemorrhagic fever, the severe form of dengue virus infection, is believed to be an immunopathological response to a secondary infection with a heterologous serotype of dengue virus. Dengue virus capsid protein-specific CD4(+) cytotoxic T-lymphocyte (CTL) clones were shown to be capable of mediating bystander lysis of non-antigen-presenting target cells. After activation by anti-CD3 or in the presence of unlabeled antigen-presenting target cells, these clones could lyse both Jurkat cells and HepG2 cells as bystander targets. Lysis of HepG2 cells suggests a potential role for CD4(+) CTL in the liver involvement observed during dengue virus infection. Three CD4(+) CTL clones were demonstrated to lyse cognate, antigen-presenting target cells by a mechanism that primarily involves perforin, while bystander lysis occurred through Fas/Fas ligand interactions. In contrast, one clone used a Fas/Fas ligand mechanism to lyse both cognate and bystander targets. Cytokine production by the CTL clones was also examined. In response to stimulation with D2 antigen, CD4(+) T-cell clones produced gamma interferon, tumor necrosis factor alpha (TNF-alpha) and TNF-beta. The data suggest that CD4(+) CTL clones may contribute to the immunopathology observed upon secondary dengue virus infections through direct cytolysis and/or cytokine production.

Identification of two epitopes on the dengue 4 virus capsid protein recognized by a serotype-specific and a panel of serotype-cross-reactive human CD4+ cytotoxic T-lymphocyte clones.

We analyzed the CD4+ T-lymphocyte response of a donor who had received an experimental live-attenuated dengue 4 virus (D4V) vaccine. Bulk culture proliferative responses of peripheral blood mononuclear cells (PBMC) to noninfectious dengue virus (DV) antigens showed the highest proliferation to D4V antigen, with lesser, cross-reactive proliferation to D2V antigen. We established CD4+ cytotoxic T-lymphocyte clones (CTL) by stimulation with D4 antigen. Using recombinant baculovirus antigens, we identified seven CTL clones that recognized D4V capsid protein. Six of these CTL clones were cross-reactive between D2 and D4, and one clone was specific for D4. Using synthetic peptides, we found that the D4V-specific CTL clone recognized an epitope between amino acids (aa) 47 and 55 of the capsid protein, while the cross-reactive CTL clones each recognized epitopes in a separate location, between aa 83 and 92, which is conserved between D2V and D4V. This region of the capsid protein induced a variety of CD4+ T-cell responses, as indicated by the fact that six clones which recognized a peptide spanning this region showed heterogeneity in their recognition of truncations of this same peptide. The bulk culture response of the donor's PBMC to the epitope peptide spanning aa 84 to 92 was also examined. Peptides containing this epitope induced proliferation of the donor's PBMC in bulk culture, but peptides not containing the entire epitope did not induce proliferation. Also, PBMC stimulated in bulk culture with noninfectious D4V antigen lysed autologous target cells pulsed with peptides containing aa 84 to 92. These results indicate that this donor exhibits memory CD4+ T-cell responses directed against the DV capsid protein and suggest that the response to the capsid protein is dominant not only in vitro at the clonal level but in bulk culture responses as well. Since previous studies have indicated that the CTL responses to DV infection seem to be directed mainly against the envelope (E) and NS3 proteins, these results are the first to indicate that the DV capsid protein is also a target of the antiviral T-cell response.

Preferential usage of T-cell receptor V beta 17 by dengue virus-specific human T lymphocytes in a donor with immunity to dengue virus type 4.

In order to better understand human T-cell responses to dengue viruses (DV), we analyzed T-cell receptor (TCR) V beta gene usage in DV-specific T lymphocytes. Peripheral blood T lymphocytes from a DV type 4 immune donor were stimulated in vitro with DV type 4 antigen, and TCR usage was examined by reverse transcriptase PCR. TCR V beta 17 was preferentially used (P = 0.020) among T cells stimulated by DV type 4 in bulk culture. Furthermore, 8 of 19 DV type 4-specific CD4+ T-cell clones established from the peripheral blood mononuclear cells expressed V beta 17 (P = 0.008). Preferential usage of TCR V alpha was not found among T cells expressing V beta 17. These results indicate that there is preferential usage of TCR V beta 17 among DV-specific T cells in this donor and suggest that T cells with certain TCRs may be important in immune responses to DV.

Immunopathologic mechanisms of dengue hemorrhagic fever and dengue shock syndrome.

Dengue virus infections are a major cause of morbidity and mortality in tropical and subtropical areas of the world. The immunopathological mechanisms that result in severe complications of dengue virus infection, i.e. dengue hemorrhagic fever (DHF), are important to determine. Primary dengue virus infections induce serotype-specific and serotype-cross-reactive, CD4+ and CD8+ memory cytotoxic T lymphocytes (CTL). In secondary infections with a virus of a different serotype from that which caused primary infections, the presence of cross-reactive non-neutralizing antibodies results in an increased number of infected monocytes by dengue virus--antibody complexes. This in turn results in marked activation of serotype cross-reactive CD4+ and CD8+ memory CTL. We hypothesize that the rapid release of cytokines and chemical mediators caused by T cell activation and by CTL-mediated lysis of dengue virus-infected monocytes triggers the plasma leakage and hemorrhage that occurs in DHF.