HLA-E-restricted recognition of cytomegalovirus-derived peptides by human CD8+ cytolytic T lymphocytes

Abstract

HLA-E-restricted T cell receptor alphabeta+ CD8+ cytolytic T lymphocytes (CTLs) exist as monoclonal expansions in the peripheral blood of some individuals. Here, we show that they recognize, with high avidity, peptides derived from the UL40 protein of different human cytomegalovirus (CMV) strains. Recognition results in the induction of cytotoxicity, IFN-gamma production and cell proliferation. Autologous cells pulsed with CMV-derived peptides become susceptible to lysis by HLA-E-restricted CTLs and induce their proliferation. The high avidity for CMV-derived peptides may explain how these cells are generated in vivo and suggest their possible role in the host defenses against CMV, a virus that evolved various mechanisms to down-regulate classical HLA class I molecules, thus escaping detection by conventional CTLs.

Fig. 1.

Different avidity in peptide recognition by NK-CTLs from different donors. Two representative NK-CTL clones derived from group 1 (GF2.1) and group 2 (KK3.2) donors were tested for cytolytic activity (in a 4-h ⁵¹Cr-release assay) (A and C) or for IFN-γ production (in an 18-h assay) (B and D) against RMA-S/HLA-E cells pulsed with graded amounts of the indicated HLA-E-binding peptides. Similar results were obtained in nine independent experiments by using clones derived from the same or different donors.

Fig. 2.

Binding of HLA-E tetramers to the TCR of NK-CTLs. NK-CTL populations from donor GF (group 1) or from donor KK (group 2), characterized by the expression of TCRVβ22 and TCRVβ16, 65% and 35%, respectively, were stained with HLA-E tetramers^PE refolded with either VMAPRTLIL (A and C) or VMAPRTLVL (B and D) peptides. Experiments were performed in the absence or presence of anti-CD94 mAb. Similar results were obtained in five independent experiments.

Fig. 3.

Peptide-induced lysis of resistant target cells by NK-CTLs. NK-CTL populations were derived from group 1 (A) and group 2 (B) donors and analyzed for their ability to lyse either autologous or resistant allogeneic B-EBV target cells (in a 4-h ⁵¹Cr-release assay). Target cells were either unpulsed or pulsed overnight at 37°C with the indicated HLA-E-binding peptides. Similar results were obtained in five independent experiments.

Fig. 4.

NK-CTLs recognize a naturally processed CMV peptide in UL40⁺ cell transfectants. NK-CTL population derived from group 2 donor KK (TCRVβ16⁺) was tested in a cytolytic assay against HEK-293T tumor cell line, either untransfected (⋄) or cotransfected (▴) with HLA-E*01033 and AD169-derived UL40 in a 4-h ⁵¹Cr-release assay. Untransfected HEK-293T cell line was also analyzed after pulsing with exogenous AD169-UL40-derived peptide (VMAPRTLIL) (○). Similar results were obtained in eight independent experiments.

Fig. 5.

NK-CTL proliferation in response to autologous APC pulsed with UL40-derived peptide (VMAPRTLIL). NK-CTL-specific expansions from donor GF (group 1; A) or from donor KK (group 2; C), obtained after priming CD8⁺ T cells with autologous APC pulsed with VMAPRTLIL peptide, were characterized by the expression of TCRVβ22 and TCRVβ16, 34% and 25%, respectively. These cell populations were tested for cytolytic activity (in a 4-h ⁵¹Cr-release assay) against RMA-S/HLA-E cells pulsed with graded amounts of the indicated peptides (B and D). Similar results were obtained in four independent experiments.