Limited T cell receptor diversity of HCV-specific T cell responses is associated with CTL escape

Abstract

Escape mutations are believed to be important contributors to immune evasion by rapidly evolving viruses such as hepatitis C virus (HCV). We show that the majority of HCV-specific cytotoxic T lymphocyte (CTL) responses directed against viral epitopes that escaped immune recognition in HCV-infected chimpanzees displayed a reduced CDR3 amino acid diversity when compared with responses in which no CTL epitope variation was detected during chronic infection or with those associated with protective immunity. Decreased T cell receptor (TCR) CDR3 amino acid diversity in chronic infection could be detected long before the appearance of viral escape mutations in the plasma. In both chronic and resolved infection, identical T cell receptor clonotypes were present in liver and peripheral blood. These findings provide a deeper understanding of the evolution of CTL epitope variations in chronic viral infections and highlight the importance of the generation and maintenance of a diverse TCR repertoire directed against individual epitopes.

Figure 1.

TCRα/β repertoire of liver-derived HCV-specific CD8⁺ T cells targeting CTL epitopes undergoing escape mutations in chronically infected chimpanzees. (A) TCRα/β rearrangements of HCV-specific, liver-derived CD8⁺ T cell clones in chimpanzee CBO603 directed against NS4₁₉₆₃. (B) TCR α/β rearrangements of HCV-specific, liver-derived CD8⁺ T cell clones in chimpanzee CH-503 directed against NS5₁₉₈₉. T cell clones with identical nucleotide sequences in TCRα and TCRβ are identified with symbols (*, ^§) at the end of the sequence.

Figure 2.

Longitudinal analysis of the TCRα/β repertoire of HCV-E2₅₈₈–specific CD8⁺ T cells before and after the detection of CTL epitope variations. (A) E2₅₈₈-specific T cells were derived from the liver of chimpanzee CH-503 7 mo after primary infection, but before the appearance of escape mutations. (B) TCRα/β rearrangement of liver-derived T cell clones generated by limiting dilution 7 yr after primary infection. (C) PBMC-derived TCRβ clonotypes 9 yr after primary infection. 67 sequences were evaluated at month 110, and 70 sequences were evaluated at month 112 after infection. Amino acid motifs within the CDR3 antigen recognition site are shown (bold). Homogenous areas within the CDR3 repertoire are indicated (boxed). Gray areas indicate nucleotide differences of clonotypes with identical amino acid motifs. T cell clones with identical nucleotide sequences in TCRα and TCRβ are identfied with symbols (*, **, ^§) at the end of the sequence.

Figure 3.

Analysis of amino acid diversity of the CDR3/joining region in HCV-specific T cell responses. Shannon entropies per amino acid site for unselected T cells (A) and CD8⁺ T cells specific for E2₅₈₈ (B) and E1₂₃₃ (C) in CH-503 are shown. (D–G) CD8⁺ T cells specific for E2₄₄₅ in CBO572 6 yr after resolution of primary infection (D) and 14 d after rechallenge with a homologous virus at the peak of the immune response (E), and for CD4⁺ T cells specific for NS3₁₂₄₈ 6 yr after resolution of primary infection (F) and 14 d after rechallenge with a homologous virus at the peak of the immune response (G). Bar graphs indicate the entropy at the respective amino acid site. Conserved amino acid residues are shown (gray bars). Amino acid residues are numbered starting with the conserved NH₂-terminal cysteine of the TCR variable region.

Figure 4.

TCRβ clonotypes in chronic infection without CTL epitope variants. TCRβ clonotypes of E1₂₃₃-specific CD8⁺ cells derived from the peripheral blood of CH-503, where no escape was detectable during chronic infection.

Figure 5.

TCR clonotypes after resolved infection, and after rechallenge with homologous virus. (A) The majority of TCRβ clonotypes of E2₄₄₅-specific CD8⁺ T cells from CBO572 are identical before and after rechallenge. The frequencies of individual clonotypes 6 yr after resolved primary infection (memory) and 14 d after rechallenge with homologous virus are shown. (B) TCRβ rearrangement and CDR3 sequences of three CD8⁺ T cell clones derived from liver or PBMCs derived from CBO572 5 yr after primary infection. (C) T cell clones show E2₄₄₅-specific cytotoxic activity as tested in chromium release assay against autologous target cells at an effector/target cell ratio of 5:1.

Figure 6.

TCR clonotypes after resolved infection, and after rechallenge with homologous virus. TCRβ clonotypes of NS3₁₂₄₈-specific CD4⁺ T cells from CBO572 before and after rechallenge. The frequencies of individual clonotypes 6 yr after resolved primary infection (memory) and 14 d after rechallenge with homologous virus are shown. *, sequences with identical CDR3 amino acid sequences, but rearrangement of different variable regions.

Figure 7.

CDR3 entropy of HCV-specific responses from chronically infected chimpanzees CBO603 and CH-503 and chimpanzee CBO572 after resolved HCV infection. Bars represent mean CDR3 entropy (±SD) of unselected T cells (gray bar), HCV-specific T cell responses associated with CTL escape (white bars), and those not associated with CTL escape (black bars).