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Characterization of the Expression Profiles of Ligands to Activating Natural Killer Cell Receptors on the HLA-Null Cell Lines K5

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Characterization of the Expression Profiles of Ligands to Activating Natural Killer Cell Receptors on the HLA-Null Cell Lines K5 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 Alexandra Tremblay-McLean Department of Experimental Medicine McGill University A thesis submitted to the Faculty of Graduate Studies and Research In fulfilment of the requirements for the degree of Master of Science © Alexandra Tremblay-McLean February 2017 1 Characterization of the expression profiles of ligands to activating Natural 2 Killer cell receptors on the HLA-null cell lines K562 and 721.221 and on 3 HIV-1 infected or non-infected CD4+ T cells 4 Alexandra Tremblay-McLean 5 Abstract 6 Natural Killer (NK) cells direct anti-viral responses through a process dependent on the 7 integration of signals received from inhibitory and activating NK receptors (iNKR and aNKR). 8 NK cells can be activated by autologous HIV-infected CD4 T cells (iCD4) to inhibit HIV 9 replication. While iNKR and the downregulation of their HLA ligands on iCD4s have been 10 investigated, the contribution of aNKR and their ligands on iCD4 to NK cell activation and 11 subsequent anti-viral responses remain unclear. Additionally, previous work from our lab showed 12 that the HLA-null cell lines 721.221 (721) and K562 activate different frequencies and functional 13 subsets of NK cells. These cell lines do not express iNKR ligands, but their aNKR ligand profile 14 may differ in a manner that explains the how they activate NK cell differentially. In this thesis, I 15 will describe experiments that characterized the aNKR profile of iCD4 and HLA null cells to 16 improve our understanding of the way in which these cells activate NK cells. Using flow 17 cytometry, I analyzed the expression of a panel of ligands to aNKR on iCD4, K562, and 721 cells. 18 This panel included ULBP-1, ULBP-2/5/6, ULBP-3, MIC-A, MIC-B, CD48, CD80, CD86, 19 CD112, CD155, ICAM-1, ICAM-2, HLA-E, HLA-F, the ligands to the aNKR NKp30, NKp44, 20 NKp46, and KIR3DS1. I also compared the expression of HLA-A2 and HLA-C in uninfected CD4 21 and iCD4, which are differentially impacted by HIV infection. I found that, while K562 and 721 22 cells shared the expression of several aNKR ligands, K562 cells were characterized by the 23 expression of ULBP-2/5/6, ULBP-3, CD112, CD155, and the ligand to NKp30 and 721 cells were 24 characterized by the expression of MIC-B, CD48, CD80, CD86, the ligand to NKp44, and HLA- 25 E. The aNKR ligand profile of 721 cells is capable of interacting with a greater breadth of aNKR 26 on NK cells than that of K562 cells, which may in part explain why 721 cells can stimulate greater 27 frequencies of NK cells. Additionally, I found that iCD4 T cells, compared to uninfected CD4 T 28 cells, trended towards expressing a higher frequency and intensity, of aNKR ligands. I also 29 observed that iCD4 that maintained CD4 expression at their surface expressed greater levels of 30 aNKR ligands than iCD4 where membrane CD4 levels were downmodulated by the HIV Nef and 31 Vpu (iCD4-). The levels of aNKR ligand expression in iCD4- were similar to those of uninfected 32 CD4 T cells for ULBP-1, ULBP-2/5/6, ULBP-3, MIC-A and CD80. In contrast, the levels of 33 aNKR ligand expression in iCD4- were below those of uninfected cells for MIC-B, CD48, CD112, 34 CD155, and ICAM-2. These findings suggest that ligands to aNKR are transiently upregulated in 35 T cells upon HIV infection and that this is followed by a downregulation in what is thought to be 36 the more productively infected iCD4-. The ligands that are downregulated to levels below those in 37 uninfected cells are of particular interest for future research, as reducing the expression of these 38 ligands and their subsequent interactions with NK cell receptors may confer a survival advantage 39 to HIV infected cells. i 40 Profils d’expression des ligands de récepteurs activateurs de cellules NK 41 sur des lignées HLA-nul et des cellules T CD4+ infectées ou non par le 42 VIH-1 43 Alexandra Tremblay-McLean 44 Abrégé 45 Les réponses antivirales dirigées par les cellules NK dépendent de l’intégration de signaux 46 transmis par les récepteurs inhibiteurs (iNKR) et activateurs (aNKR) qu’elles expriment. Les 47 cellules T CD4+ infectées par le VIH-1 (iCD4) sont capables d’activer les cellules NK qui vont 48 ensuite inhiber la réplication du virus. Alors que l’impact des iNKR et de leurs ligands HLA 49 (exprimés par les iCD4) sur la réplication virale est relativement bien connu, la contribution des 50 aNKR et leurs ligands demeure moins bien compris. De récents travaux menés au laboratoire ont 51 montré que les lignées cellulaires K562 et 721.221 activaient de manière distincte les cellules NK 52 tant au point de vu qualitatif que quantitatif. Dans ce travail, nous avons choisi d’étudier le profil 53 d’expression des ligands de aNKR sur les iCD4 et les lignées cellulaires HLA-nul afin de mieux 54 comprendre comment ces ligands stimulent les cellules NK. Pour ce faire, nous avons analysé par 55 cytométrie de flux l’expression d’un panel de ligands de aNKRs sur les iCD4, les K562 et les 56 721.221. Ce panel inclut ULBP-1, ULBP-2/5/6, ULBP-3, MIC-A, MIC-B, CD48, CD80, CD86, 57 CD112, CD155, ICAM-1, ICAM-2, HLA-E, HLA-F, les ligands de NKp30, NKp44, NKp46, 58 KIR3DS1 et HLA-A2 / HLA-C qui sont des ligands d’iNKR exprimés par les cellules NK. Nos 59 résultats montrent que les cellules K562 et 721 expriment des niveaux similaires de plusieurs 60 ligands de aNKR. Toutefois, les K562 se caractérisent par une expression plus importante de 61 ULBP-2/5/6, ULBP-3, CD112, CD155 et du ligand de NKp30; alors que les 721 se caractérisent 62 par une expression plus marquée de MIC-B, CD48, CD80, CD86, du ligand de NKp44 et de HLA- 63 E. Globalement, les cellules 721 expriment davantage de ligands susceptibles d’interagir avec les 64 aNKR présents sur les cellules NK, ce qui pourrait expliquer leur meilleure capacité à stimuler ces 65 cellules. Par ailleurs, nous avons observé que les cellules T CD4+ infectées par le VIH avaient 66 tendance à exprimer des niveaux plus élevés de ligands de aNKR que les cellules non-infectées, et 67 ceci en terme de fréquence de cellules positives et d’intensité moyenne de fluorescence. Nous 68 avons également observé que l’expression de ces ligands était plus élevée sur les cellules infectées 69 qui ont gardé le CD4 à la membrane (iCD4+), comparativement aux cellules infectées qui n’ont 70 plus de CD4 membranaire (iCD4-), suite probablement à son internalisation par la protéine Nef du 71 VIH. Une analyse plus détaillée montre que les iCD4- présentent des niveaux de ULBP-1, ULBP- 72 2/5/6, ULBP-3, MICA et CD80 semblables aux cellules non-infectées, et des niveaux de MICB, 73 CD48, CD112, CD155 et ICAM-2 encore plus réduit que les cellules non-infectées. L’ensemble 74 de nos données suggère un modèle selon lequel l’expression des ligands de aNKRs serait 75 transitoirement augmentée à la surface des cellules T CD4+ lors de l’infection précoce par le VIH, 76 puis réduite sur les cellules infectées de manière productive, à l’instar du CD4. L’impact de 77 l’infection VIH sur l’expression des ligands de aNKR est d’un intérêt particulier pour la recherche 78 future dans la mesure ou la réduction de leur expression membranaire et, par conséquent, la 79 diminution de leur capacité à activer les cellules NK pourrait accorder un avantage de survie aux 80 cellules productrices de virus qui échapperaient à la lyse par les cellules NK. 81 ii 82 Acknowledgements 83 First and foremost, I would like to acknowledge my supervisor Dr. Nicole Bernard, who 84 provided the seeds that would germinate into the research presented here and also the 85 environment, support, and guidance required to bring those ideas to fruition. Additionally, I owe 86 a debt of gratitude to many members of our lab, both past and present. First, to Drs. Gamze 87 Isitman and Irene Lisovsky, who provided vital training and help with the flow cytometric 88 aspects of this work. To Drs. Sandrina DaFonseca and Franck Dupuis for helping with various 89 troubleshooting issues and for help refining this work. To our technicians, Xiaoyan Ni and 90 Tsoarello Mabanga, who worked tirelessly to procure reagents and the cryopreserved cell 91 samples that were the foundation of this work. Finally, to all the lab members not listed here who 92 helped along the way. 93 94 Many thanks to Dr. Galit Alter for the kind gift of our 721.221 cell line and to our 95 colleagues responsible for maintaining and organizing the HIV seronegative donor cohorts and 96 establishing the infrastructure required to provide us with leukapheresis samples. In particular, 97 I’d like to thank Dr. Julie Bruneau, Dr. Betrand Lebouché, Dr. Jean Pierre Routy, Dr Cecile 98 Tremblay and the hard-working nurses Josée Girouard and Pascale Arlotto. Finally, and with 99 enormous gratitude, I would like to acknowledge those who generously donated samples and 100 cells, without whom none of this research would have been possible. These individuals provided 101 a constant and crucial reminder of the real people that I hope one day my research may positively 102 impact.
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