Differential functional avidity of dengue virus-specific T-cell clones for variant peptides representing heterologous and previously encountered serotypes

Abstract

Proinflammatory cytokines secreted by memory CD8+ and CD4+ T cells are thought to play a direct role in the pathogenesis of dengue virus infection by increasing vascular permeability and thereby inducing the pathophysiologic events associated with dengue hemorrhagic fever and dengue shock syndrome. Severe disease is frequently observed in the setting of secondary infection with heterologous dengue virus serotypes, suggesting a role for cross-reactive memory T cells in the immunopathogenesis of severe disease. We used a large panel of well-characterized dengue virus-specific CD8+ T-cell clones isolated from Pacific Islanders previously infected with dengue virus 1 to examine effector memory function, focusing on a novel dominant HLA-B*5502-restricted NS5(329-337) epitope, and assessed T-cell responses to stimulation with variant peptides representing heterologous serotypes. Variant peptides were differentially recognized by dengue virus 1-specific effector CD8+ cytotoxic T lymphocytes (CTL) in a heterogeneous and clone-specific manner, in which cytolytic function and cytokine secretion could be enhanced, diminished, or abrogated compared with cognate peptide stimulation. Dengue virus-specific CTL stimulated with cognate and variant peptides demonstrated a cytokine response hierarchy of gamma IFN (IFN-gamma) > tumor necrosis factor alpha (TNF-alpha) > interleukin-2 (IL-2), and a subset of clones also produced IL-4 and IL-6. Individual clones demonstrated greater avidity for variant peptides representing heterologous serotypes, including serotypes previously encountered by the subject, and IFN-gamma and TNF-alpha secretion was enhanced by stimulation with these heterologous peptides. Altered antiviral T-cell responses in response to stimulation with heterologous dengue virus serotypes have implications for control of virus replication and for disease pathogenesis.

FIG. 1.

The Den-1 NS5_329-337 CD8⁺ T-cell epitope is restricted by HLA-B*5502. Responses to the epitope were CD8 dependent; depletion of CD8⁺ T cells with antibody-labeled magnetic beads abrogated the IFN-γ ELISPOT response, and only the PBMC and CD8⁺ fractions were positive (A). T-cell lines generated by stimulation of PBMC collected up to 5 years after infection with ELISPOT-mapped cognate peptide were cytolytic. Den-1 NS5_329-337-specific T cells lysed autologous peptide-pulsed BLCL in a 5-hour ⁵¹Cr-release assay. All three subjects who responded to the epitope shared the molecule HLA B*5502, which was confirmed to be the restricting element in mismatched target cytotoxicity assays. Target cells were pulsed with cognate peptide and were either autologous BLCL or donor BLCL which matched at only one allele. Strong lytic responses were only seen for the autologous cell line and for the donor cell line expressing HLA B55 (B). A representative experiment shows responses measured for subject 3.

FIG. 2.

Den-1-infected individuals mount effector T-cell responses to variant epitope peptides representing heterologous serotypes. Analysis of polyclonal T-cell populations indicates there is a high degree of cross-reactivity between cognate Den-1 KP9 NS5_329-337 epitope peptide and variant peptides representing heterologous dengue viruses. High-avidity responses were measured by direct ex vivo IFN-γ ELISPOT of whole-fraction PBMC (A) and for T-cell lines derived by stimulation of PBMC with cognate NS5_329-337 KPWDVIPMV peptide (B) and tested in ⁵¹Cr release assays, in which target cells were autologous BLCL pulsed with NS5_329-337 epitope peptide KP9 (▪) or variant peptides representing heterologous dengue viruses D2-1 (▴), D2-2 (▾), D2-3 (⧫), D2-4 (•), and D3 (□). Similar data were obtained for subjects 2, 3, and 7; representative experiments from subject 7 are shown.

FIG. 3.

Differential functional avidities of Den-1-specific T-cell clones for altered peptide ligands representing heterologous and previously encountered serotypes. Den-1 NS5_329-337-specific CD8⁺ T-cell clones were incubated with ⁵¹Cr-labeled autologous transformed B cells pulsed with cognate D1 epitope peptide KP9 (▪) and variant peptides D2-1 (▴), D2-2 (▾), D2-3 (⧫), D2-4 (•), and D3 (▪). Peptide titration curves suggest that functional avidity for heterologous serotypes may be enhanced compared to responses for cognate epitope peptides (A). Secretion of the proinflammatory cytokines IFN-γ (B) and TNF-α (C) was also differentially altered when clones were stimulated with altered peptide ligands, particularly D2-2 (▾) and D3 (□), compared with cognate KP9 Den-1 peptide (□). Clones 10G4 and 2C9 were isolated from subject 2; clones 8H2, 1C12, and 9A2 were isolated from subject 3.

FIG. 3.

Differential functional avidities of Den-1-specific T-cell clones for altered peptide ligands representing heterologous and previously encountered serotypes. Den-1 NS5_329-337-specific CD8⁺ T-cell clones were incubated with ⁵¹Cr-labeled autologous transformed B cells pulsed with cognate D1 epitope peptide KP9 (▪) and variant peptides D2-1 (▴), D2-2 (▾), D2-3 (⧫), D2-4 (•), and D3 (▪). Peptide titration curves suggest that functional avidity for heterologous serotypes may be enhanced compared to responses for cognate epitope peptides (A). Secretion of the proinflammatory cytokines IFN-γ (B) and TNF-α (C) was also differentially altered when clones were stimulated with altered peptide ligands, particularly D2-2 (▾) and D3 (□), compared with cognate KP9 Den-1 peptide (□). Clones 10G4 and 2C9 were isolated from subject 2; clones 8H2, 1C12, and 9A2 were isolated from subject 3.

FIG. 3.

Differential functional avidities of Den-1-specific T-cell clones for altered peptide ligands representing heterologous and previously encountered serotypes. Den-1 NS5_329-337-specific CD8⁺ T-cell clones were incubated with ⁵¹Cr-labeled autologous transformed B cells pulsed with cognate D1 epitope peptide KP9 (▪) and variant peptides D2-1 (▴), D2-2 (▾), D2-3 (⧫), D2-4 (•), and D3 (▪). Peptide titration curves suggest that functional avidity for heterologous serotypes may be enhanced compared to responses for cognate epitope peptides (A). Secretion of the proinflammatory cytokines IFN-γ (B) and TNF-α (C) was also differentially altered when clones were stimulated with altered peptide ligands, particularly D2-2 (▾) and D3 (□), compared with cognate KP9 Den-1 peptide (□). Clones 10G4 and 2C9 were isolated from subject 2; clones 8H2, 1C12, and 9A2 were isolated from subject 3.

FIG. 4.

Den-1 NS5_329-337-specific CD8⁺ T-cell clones demonstrate a cytokine phenotype hierarchy of IFN-γ > TNF-α > IL-2 when stimulated with cognate and altered peptide ligands. Cell lines were incubated with peptide-pulsed autologous BLCL at a ratio of 10:1 for 5 h, and harvested supernatants were tested for cytokine production by flow cytometric cytokine bead array. Seventeen clones were analyzed. Results are expressed as the percent total clones analyzed secreting the relevant cytokines. All clones produced IFN-γ on stimulation with cognate peptide, and responses were variably reduced in response to variant peptide ligands. The Tc2 cytokines IL-4 and IL-6 were secreted by a subset of clones which also produced Tc1 cytokines.