Altered expression of ligands for the NKG2D and DNAM-1 activating receptors during HIV-1 infection

Background

Human cells may respond to viral infection or other stress by expressing on their membrane ligands for activating receptors present on cytotoxic NK and T cells, such as NKG2D and DNAM-1 receptors, thus eliciting recognition and elimination by the immune system. Work from our laboratory has shown that, upon infection with HIV-1, CD4⁺ T cells over-express ligands for NKG2D (MICA/B and ULBP2) and DNAM-1 (PVR), hence becoming susceptible to NKG2D-and DNAM-1-mediated lysis by NK cells. The cell-surface expression of activating ligands, however, is down-regulated by the HIV-1 Nef and Vpu proteins, a phenomenon that protects infected cells from cytotoxic responses to some extent. Here we further investigated the dual regulation of activating ligands by HIV-1 focusing on viral and cellular factors involved in up-regulation of ligands expression and on the capacity of HIV-infected cells to release soluble ligands in the extracellular environment.

Materials and methods

Jurkat T cells and primary CD4+ T lymphocytes were transduced with HIV-1 or individual viral genes (wt or mutated). Ligands expression was analyzed by measuring mRNA, cell-surface and total protein levels. Activation of the DNA Damage Response (DDR) pathway and cell cycle profile were also analyzed. Soluble ligands were measured by ELISA in the medium of in vitro infected cells as well as in the plasma of HIV-infected patients. The impact of soluble ligands on the expression and function of their cognate receptor was investigated by FACS-based immunofluorescence analysis and cytotoxicity assays.

Results

Results showed that the HIV-1 Vpr protein increases cell-surface and total PVR levels acting at a post-transcriptional level. As reported previously for NKG2D ligands, PVR was up-regulated by Vpr via activation of the ATR kinase that triggers the DDR pathway and G₂ arrest. Increased expression of PVR and NKG2D ligands correlated with their higher release in HIV-infected T cell cultures. Moreover, treatment-naïve HIV-infected patients displayed increased plasma levels of soluble MICA and ULBP2 and reduced NKG2D expression on NK and CD8+ T cells. However, uptake of antiretroviral therapy (ART) resulted in the drop of soluble NKG2D ligands and recovery of NKG2D expression. Finally, we found that NKG2D ligands in patients’ plasma down-regulated NKG2D on NK and CD8+ T cells and impaired NKG2D-mediated cytotoxicity of NK cells.

Conclusions

Attempts to boost the DDR-mediated up-regulation of NKG2D and DNAM-1 ligands represent novel attractive approaches to improve recognition and elimination of HIV-infected cells by the immune system. By promoting the release of soluble ligands, HIV-1 may simultaneously attenuate their expression on infected cells and down-regulate cognate receptors on effector cells, thus subverting immuno-surveillance against HIV-1 and opportunistic infections, but ART has the potential to avoid such immune dysfunction.