Dissertation

Characterization of the Expression Profiles of Ligands to Activating Natural Killer Cell Receptors on the HLA-Null Cell Lines K562 and 721.221 and on HIV-1 Infected or Non-Infected CD4+T Cells

Tremblay-McLean, Alexandra

2017

Overview

Natural Killer (NK) cells direct anti-viral responses through a process dependent on the integration of signals received from inhibitory and activating NK receptors (iNKR and aNKR). NK cells can be activated by autologous HIV-infected CD4 T cells (iCD4) to inhibit HIV replication. While iNKR and the downregulation of their HLA ligands on iCD4s have been investigated, the contribution of aNKR and their ligands on iCD4 to NK cell activation and subsequent anti-viral responses remain unclear. Additionally, previous work from our lab showed that the HLA-null cell lines 721.221 (721) and K562 activate different frequencies and functional subsets of NK cells. These cell lines do not express iNKR ligands, but their aNKR ligand profile may differ in a manner that explains the how they activate NK cell differentially. In this thesis, I will describe experiments that characterized the aNKR profile of iCD4 and HLA null cells to improve our understanding of the way in which these cells activate NK cells. Using flow cytometry, I analyzed the expression of a panel of ligands to aNKR on iCD4, K562, and 721 cells. This panel included ULBP-1, ULBP-2/5/6, ULBP-3, MIC-A, MIC-B, CD48, CD80, CD86, CD112, CD155, ICAM-1, ICAM-2, HLA-E, HLA-F, the ligands to the aNKR NKp30, NKp44, NKp46, and KIR3DS1. I also compared the expression of HLA-A2 and HLA-C in uninfected CD4 and iCD4, which are differentially impacted by HIV infection. I found that, while K562 and 721 cells shared the expression of several aNKR ligands, K562 cells were characterized by the expression of ULBP-2/5/6, ULBP-3, CD112, CD155, and the ligand to NKp30 and 721 cells were characterized by the expression of MIC-B, CD48, CD80, CD86, the ligand to NKp44, and HLA-E. The aNKR ligand profile of 721 cells is capable of interacting with a greater breadth of aNKR on NK cells than that of K562 cells, which may in part explain why 721 cells can stimulate greater frequencies of NK cells. Additionally, I found that iCD4 T cells, compared to uninfected CD4 T cells, trended towards expressing a higher frequency and intensity, of aNKR ligands. I also observed that iCD4 that maintained CD4 expression at their surface expressed greater levels of aNKR ligands than iCD4 where membrane CD4 levels were downmodulated by the HIV Nef and Vpu (iCD4-). The levels of aNKR ligand expression in iCD4- were similar to those of uninfected CD4 T cells for ULBP-1, ULBP-2/5/6, ULBP-3, MIC-A and CD80. In contrast, the levels of aNKR ligand expression in iCD4- were below those of uninfected cells for MIC-B, CD48, CD112, CD155, and ICAM-2. These findings suggest that ligands to aNKR are transiently upregulated in T cells upon HIV infection and that this is followed by a downregulation in what is thought to be the more productively infected iCD4-. The ligands that are downregulated to levels below those in uninfected cells are of particular interest for future research, as reducing the expression of these ligands and their subsequent interactions with NK cell receptors may confer a survival advantage to HIV infected cells.

• Publisher: ProQuest Dissertations & Theses
• Subject: Binding sites / Cytotoxicity / HIV / Human immunodeficiency virus / Lymphocytes / Medicine / Molecular biology / Virology / Viruses
• ISBN: 9798582588771