Abstract
Human cytomegalovirus (HCMV) infection promotes the differentiation and persistent expansion of a mature NK cell subset, which displays high surface levels of the activating CD94/NKG2C NK cell receptor, together with additional distinctive phenotypic and functional features. The mechanisms underlying the development of adaptive NK cells remain uncertain but some observations support the involvement of a cognate interaction of CD94/NKG2C with ligand(s) displayed by HCMV-infected cells. To approach this issue, the heterodimer and its adaptor (DAP12) were expressed in the human Jurkat leukemia T cell line; signaling was detected by transfection of a reporter plasmid encoding for Luciferase (Luc) under NFAT/AP1-dependent control. Engagement of the receptor by solid-phase bound CD94- or NKG2C-specific monoclonal antibodies (mAbs) triggered Luc expression. Moreover, reporter activation was detectable upon interaction with HLA-E+ 721.221 (.221-AEH) cells, as well as with 721.221 cells incubated with synthetic peptides, which stabilized surface expression of endogenous HLA-E; the response was specifically antagonized by soluble NKG2C- and HLA-E-specific mAbs. By contrast, activation of Jurkat-NKG2C+ was undetectable upon interaction with Human Fetal Foreskin Fibroblasts (HFFF) infected with HCMV laboratory strains (i.e., AD169, Towne), regardless of their differential ability to preserve surface HLA-E expression. On the other hand, infection with two clinical isolates or with the endotheliotropic TB40/E strain triggered Jurkat-NKG2C+ activation; yet, this response was not inhibited by blocking mAbs and was independent of CD94/NKG2C expression. The results are discussed in the framework of previous observations supporting the hypothetical existence of specific ligand(s) for CD94/NKG2C in HCMV-infected cells.
Highlights
Inhibitory receptors specific for MHC class I molecules with immunoreceptor tyrosine-based inhibitory motifs play a key role in preventing NK cell responses against normal autologous cells
NKG2C and DAP12 were stably transduced in an available CD94-transfected human Jurkat leukemia T cell line
The rationale for using a human parental cell line was based on our previous experience with heterologous CD94/NKG2C+ rat basophilic leukemia cells (RBL), which failed to respond to receptor engagement by HLA-E+ .221-AEH cells
Summary
Inhibitory receptors specific for MHC class I molecules with immunoreceptor tyrosine-based inhibitory motifs play a key role in preventing NK cell responses against normal autologous cells. The hypothesis that MHC-specific activating NKR may contribute to the innate response against pathogens was supported by the evidence that Ly49H interacts with the MHC class I-related murine cytomegalovirus glycoprotein m157, triggering NK cell effector functions and the development of a memorylike response that confers resistance against the viral infection in some mice strains [3,4,5] With this remarkable exception, no formal proof has been far obtained supporting the involvement of other activating KIR, NKG2, or Ly49 receptors in direct recognition of pathogen molecules [6]. Expansions of NKG2C+ NK cells have been reported in the context of other infections [18,19,20,21], the effect appears restricted to individuals coinfected by HCMV, suggesting that it is induced by this herpes virus, being potentially amplified along the immune response to other pathogens
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