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. Considering their contributions, this work is dedicated to them. 103 104 105 106 107 108 109
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111 Table of Contents
112 Abstract…………………………………………………………………………………………………...... i
113 Abrégé……………………………………………………………………………………………………... ii
114 Acknowledgements……………………………………………………………………………………...... iii
115 Table of contents………………………………………………………………………………...... iv
116 Abbreviations…………………………………………………………………………………………….. vii
117 List of Tables…………………………………………………………………………………………..…... x
118 List of Figures…………………………………………………………………………………...... xi
119 Chapter 1: Introduction and Literature Review…………………………………………….... 1
120 1.1: Rationale………………………………………………………………………………….…………... 2
121 1.2.: Objectives…………………………………………………………………………………….…….... 4
122 1.3: History of the HIV pandemic………………………………………………………………….……... 6
123 1.3.2: Viral structure………………………………………………………………………………….… 7
124 1.3.3: Kinetics of infection……………………………………………………………………………... 8
125 1.3.4: HIV immunopathogenesis………………………………………………………………....….... 11
126 1.3.5: Discovery and function of NK cells………………………………………………………….… 13
127 1.3.6: NK cells in HIV………………………………………………………………………….....…... 15
128 1.4: NK cell licensing and activation…………………………………………………………………..... 16
129 1.4.2: Models for the study of NK cell activation………………………………....………………...... 18
130 1.5: Characterization of activating NK cell receptors and their ligands…………………………....….... 19
131 1.5.2: NKG2 receptors………………………………………………………………………...……... 19
132 1.5.3: Natural cytotoxicity receptors……………………………………………………………...... 23
133 1.5.4: Additional activating receptors……………………………………………………...……….... 26
134 Linker Paragraph………………………………………………………………………………...……..... 31 iv
135 Chapter 2: Manuscript 1 – The HLA-null cell lines K562 and 721.221 express different ligand 136 profiles for activating NK cell receptors …………………………………………………….... 32
137 Title page………………………………………………………………………………………..……...… 33
138 2.1: Author contributions and acknowledgements…………………………………………...... ….…… 34
139 2.2: Abstract…………………………………………………………………………………….…….... 35
140 2.3: Introduction………………………………………………………………………………….…...... 36
141 2.4: Methods………………………………………………………………………………………….… 40
142 2.4.1: Origin and preparation of K562 and 721 cell lines…………………………………………… 40
143 2.4.2: Antibody staining and acquisition……………………………………………………………. 40
144 2.4.3: Statistical analysis……………………………………………………………………………. 42
145 2.5: Results……………………………………………………………………………………………... 43
146 2.5.1: Comparison of the frequencies of K562 and 721 expressing ligands to aNKR…………….... 43
147 2.5.2: Comparison of the expression score for ligands to aNKR on the K562 and 721 HLA-null cell 148 lines ……………………………………………………………………………………………….... 44
149 2.6: Discussion…………………………………………………………………………………………. 46
150 2.7: Figure Legends………………………………………………………………………………….…. 51
151 2.8: References……………………………………………………………………………………….… 54
152 Linker Paragraph……………………………………………………………………………………….… 58
153 Chapter 3: Manuscript 2 – Expression profiles of ligands for activating NK cell receptors on 154 HIV infected and uninfected CD4+ T cells………………………………………………….… 59
155 Title page…………………………………………………………………………………………….....… 60
156 3.1: Author contributions and acknowledgements……………………………………………………... 61
157 3.2: Abstract………………………………………………………………………………………….… 62
158 3.3: Introduction………………………………………………………………………………………... 63
159 3.4: Methods……………………………………………………………………………………………. 66
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160 3.4.1: Ethics statement………………………………………………………………………….…… 66
161 3.4.2: Study population…………………………………………………………………………….... 66
162 3.4.3: CD4+ T cell isolation………………………………………………………………………….. 66
163 3.4.4: HIV infection………………………………………………………………………………..... 67
164 3.4.5: Antibody staining and acquisition……………………………………………………….…… 67
165 3.4.6: Statistical analysis……………………………………………………………………...…….. 69
166 3.5: Results………………………………………………………………………………………...... 70
167 3.5.1: CD4+ T cell infection characteristics……………………………………….………….……... 70
168 3.5.2: Comparison of the expression profiles of ligands to aNKR on HIV-infected CD4+ T cells that 169 maintain or not cell surface expression of CD4………………………………………………….. 71
170 3.5.3: Comparison of the frequencies of HIV iCD4 and unCD4 T cells expressing ligands to aNKRs 171 ………………………………………………………………………………………………….... 71
172 3.5.4: Comparison of the aNKR ligand expression intensity on iCD4 and unCD4 T cells…………. 72
173 3.5.5: Expression profiles of HLA-E, HLA-A2, and HLA-C of iCD4 and unCD4……………….... 73
174 3.6: Discussion………………………………………………………………………………………..... 74
175 3.7: Figure Legends………………………………………………………………………………….…. 80
176 3.8: References…………………………………………………………………………………..……... 92
177 Chapter 4: Discussion………………………………………………………………………...... 97
178 4.1: Contributions of this work to the field………………………………………………………...... 98
179 4.2: Modulation of cell-surface ligands by HIV Nef………………………………………………….. 100
180 4.3: Limitations and future directions…………………………………………………………………. 105
181 4.4: Conclusion………………………………………………………………………………….…….. 108
182 Appendix A: Antibodies used in the analysis of ligand expression …………………...…………...... 109
183 Appendix B: Panel of the ligands studied and their respective activating NK cell receptors...... 110
184 References…………………………………………………………………………………………...... 111 vi
185 Abbreviations ER: Endoplasmic reticulum
186 Ab: Antibody ERK: Extracellular signal-regulated kinase
187 ADCC: Antibody-dependent cellular ERM: Ezrin/radixin/moesin
+ 188 cytotoxicity euCD4: HIV uninfected CD4 T cells that
189 AIDS: Acquired immune deficiency were exposed to virus
190 syndrome FBS: Fetal bovine serum
191 ARS: Acute retroviral syndrome Grb2: Growth factor receptor-bound protein
192 aNKR: Activating natural killer cell receptor 2
193 Bp: Base pair HA: Viral hemagglutinin
194 CA: Capsid HAART: Highly-active antiretroviral
195 CML: Chronic myelogenous leukemia therapy
196 CMV: Cytomegalovirus HLA: Human leukocyte antigen
197 CSK: C-terminal Src kinase HIV: Human immunodeficiency virus
198 CTL: Cytotoxic lymphocyte ICAM: Intercellular adhesion molecule
+ 199 CTLA-4: Cytotoxic T-lymphocyte- iCD4: HIV-infected CD4 T cells
+ + 200 associated protein 4 iCD4 : HIV-infected CD4 T cells that
201 DAP: DNAX-activating protein maintain CD4 at their surface
- + 202 DC: Dendritic cell iCD4 : HIV-infected CD4 T cells with
203 DNA: Deoxyribonucleic acid downregulated surface CD4
T 204 DNAM-1: DNAX accessory molecule-1 iCD4 : Total HIV-infected CD4 T cells,
205 dPBS: Dulbecco’s phosphate buffered saline regardless of surface CD4 expression
206 dsDNA: Double-stranded DNA IFN: Interferon
207 EBV: Epstein-Barr virus Ig: Immunoglobulin vii
208 iKIR: inhibitory KIR Nef: Negative regulatory factor
209 IL: Interleukin NK: Natural killer
210 ILC: Innate lymphoid cell NKG2: Natural-killer group 2 member
211 IN: Integrase receptors
212 iNKR: Inhibitory natural killer cell receptor NTB-A: NK-T-B cell antigen
213 ITAM: Immunoreceptor tyrosine-based PBMC: Peripheral blood mononuclear cell
214 activating motif p-ERK: RNA-like endoplasmic reticulum
215 ITIM: Immunoreceptor tyrosine-based kinase
216 inhibitory motif PHA: Phytohemagglutinin
217 ITSM: Immunoreceptor tyrosine-based PIC: Pre-integration complex
218 switch motif PI3K: Phosphoinositide kinase-3
219 Kb: Kilobases pNK: Peripheral blood NK cell
220 KIR: Killer immunoglobulin-like receptor PR: protease
221 LFA-1: Lymphocyte function-associated Pyk2: Protein tyrosine kinase 2-β
222 antigen-1 RAET1: Retinoic acid early transcript 1
223 MA: Matrix protein Rev: Regulator of expression of virion
224 MAPK: Mitogen-activated protein kinase protein
225 MFI: Mean fluorescence intensity rhIL-2: Recombinant human interleukin-2
226 MHC: Major histocompatibility complex RNA: Ribonucleic acid
227 MIC: MHC class-I-chain-related protein RT: Reverse transcriptase
228 mRNA: Messenger ribonucleic acid R10: RPMI 1640 medium supplemented
229 NC: Nucleocapsid with 10% FBS; 2mM L-glutamine;
230 NCR: Natural cytotoxicity receptor
viii
100IU/mL penicillin; 100mg/mL Vpu: Viral protein unique
streptomycin Zap-70 : Zeta-chain-associated protein
231 SAP: SLAM-associated protein kinase 70
232 SC: Drosophila Schneider cell 3DL1: KIR3DL1
233 SHIP: SH2 domain-containing inositol 5'- 3DS1: KIR3DS1
234 phosphatase 721: 721.221 HLA-null cell line
235 SHP: Src homology region 2 domain-
236 containing phosphatase
237 SIV: Simian immunodeficiency virus
238 SLAM: Signaling lymphocyte activation
239 molecule
240 ssRNA: Single-stranded RNA
241 Syk: Spleen tyrosine kinase
242 Tat: Transactivator of transcription
243 TIGIT: T cell immunoreceptor with
244 immunoglobulin and ITIM motifs
245 ULBP: UL16-binding protein ligands
246 unCD4: HIV-uninfected CD4+ T cells
247 Vav1: Vav guanine nucleotide exchange
248 factor-1
249 Vif: Viral infectivity factor
250 VL: Viral load
251 Vpr: Viral protein R
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252 List of Tables
253 Chapter 3: Manuscript 2 - Expression profiles of ligands for activating NK cell receptors 254 on HIV infected and uninfected CD4+ T cells.
255 Table 1: Study population HLA genotypes………………………………………………….………….. 83
256 Table 2: Description of the aNKR ligands studied…………………………………………………...… 84
257 Appendix A: Antibodies used in the analysis of aNKR ligand expression……..………………………... 109
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271
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272 List of Figures
273 Chapter 1: Introduction and Literature Review
274 Figure 1: Schematic diagram of the HIV virion and genome…………………………………………..... 8
275 Figure 2: HIV replication cycle………………………………………………………...... 11
276 Figure 3: NKG2 receptor signaling………………………………………………………...... … 20
277 Figure 4: NKG2D ligands………………………………………………………………….…………… 21
278 Figure 5: Structure of natural cytotoxicity receptors…………………………………………………… 25
279 Chapter 2: Manuscript 1 - The HLA-null cell lines K562 and 721.221 express different ligand 280 profiles for activating NK cell receptors.
281 Figure 1: Different frequencies of K562 and 721 express ligands to activating NK cell receptors...... 52
282 Figure 2: K562 and 721 differ in the expression score of their ligands to activating NK cell receptor…...53
283 Chapter 3: Expression profiles of ligands for activating NK cell receptors on HIV infected 284 and uninfected CD4+ T cells.
285 Figure 1: Gating strategy………………………….……………………………………………………. 85
286 Figure 2: Frequencies of iCD4+ vs iCD4- expressing aNKR ligands……………….………………..… 86
287 Figure 3: Frequencies of all T cell subsets expressing aNKR ligands………………………………….. 87
288 Figure 4: Mean fluorescence intensity of aNKR ligand expressing T cell subsets……………………... 88
289 Figure 5: Expression profile of the HLA molecules HLA-E, HLA-A2, and HLA-C on CD4+ T cell 290 subsets………………………………………………………………………………………….……... 89
291 Supplemental Figure 1: NK cells preferentially target HIV-infected CD4 T cells that conserve cell-surface 292 CD4 expression…………………………………………………………………………...………….... 90
293 Supplemental Figure 2: The frequency and per-cell expression of ICAM-1 is correlated with HIV- 294 infection on all infected T cell subsets…………………………………………………………………. 91
295 Appendix B: Panel of the ligands studied and their respective activating NK cell receptors …………... 110
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296 Chapter 1: Introduction and Literature Review
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318 1.1: Rationale
319 Natural Killer (NK) cells are a subset of lymphocytes that are crucial mediators of innate
320 immune responses against stressed or otherwise damaged, virally-infected, and transformed
321 cells. NK cells can directly kill target cells through the release of cytotoxic granules and
322 secretion of cytokines and chemokines. They can utilize their secretory abilities to interact with
323 T and B cells, effectively bridging the innate and adaptive immune systems (1). Activation of
324 NK cells by targets cells encountered is a process that depends on the integration of signals
325 received through inhibitory and activating NK receptors (iNKRs and aNKRs) (2, 3). NK cells
326 can be activated through abrogation of inhibitory signaling through iNKRs if they also receive
327 activating signals through aNKRs. A variety of cell types can be used in vitro or ex vivo for NK
328 cell stimulation assays to better understand the complex process of their activation. For example,
329 HLA-null cell lines are frequently used as NK cell stimuli. Human leukocyte antigen (HLA)-null
330 cells do not express classical major histocompatibility complex (MHC)-I HLA-A, -B, or -C,
331 which are ligands for iNKRs. They are therefore unable to engage iNKRs on NK cells,
332 abrogating inhibitory signaling through these receptors, but still express ligands for aNKR and
333 can stimulate NK activation. Previous work by our group comparing NK cell stimulation by the
334 HLA-null cell lines K562 and 721.221 (721) found that they stimulated NK cells differentially in
335 terms of secretion of the cytokine IFN-γ, the chemokine CCL4, and expression of the
336 degranulation marker CD107a (4). Additionally, 721 cells activated a greater fraction of NK
337 cells, compared to K562 cells. Although the mechanisms underlying these differences are
338 currently unclear, in the absence of ligands for iNKRs, examining the expression profiles of
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339 ligands for aNKRs expressed by K562 and 721 is a promising way to address this knowledge gap
340 and further elucidate the ways in which NK cells can be activated.
341 HIV-infected CD4 T cells (iCD4s), which express reduced cell surface levels of the
342 iNKR ligands HLA-A and -B, can also stimulate NK cells ex vivo and have been shown to
343 activate NK cells to inhibit HIV-1 replication in autologous iCD4s (5). While the role of HLA-A
344 and -B downregulation in stimulating NK cells has been studied, it is unclear how ligands for
345 aNKRs contribute to NK cell activation. It is plausible that HIV infection can modulate aNKR
346 ligand expression in a similar manner to its noted effects on classical HLA expression,
347 subsequently influencing NK cell activation and anti-HIV responses. Recently, the RV144 Thai
348 trial demonstrated that a vaccine regimen that combined an ALVAC HIV vaccine prime and
349 AIDSVAX B/E vaccine boost could provide protection against subsequent HIV challenge (6).
350 Although the protection afforded was modest, this marked the first successful vaccine trial and
351 confirmed that development of an effective anti-HIV vaccine is possible. Secondary analyses of
352 samples obtained during the trial highlighted NK cell-mediated antibody-dependent cellular
353 cytotoxicity as a correlate of protection (7). These findings help emphasize the importance of a
354 thorough understanding of NK cell activation and NK-cell mediated anti-HIV responses in the
355 design of future anti-HIV vaccines and treatment. The work presented here analyses the aNKR
356 ligand expression profiles of HIV-infected and uninfected CD4+ T cells, with goal of clarifying
357 their different abilities to trigger NK cell activation and anti-HIV responses.
358
359
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360 1.2: Objectives
361 Following our previous observation that the two HLA-null cell lines K562 and 721
362 activated different frequencies of total NK cells and stimulated different functional responses, we
363 were interested in investigating (i) whether K562 and 721 possessed different aNKR ligand
364 expression profiles that could explain the differences observed and (ii) whether HIV-1-infected
365 CD4+ T cells, which are also used ex vivo to stimulate NK cells, might also influence NK
366 activation through an aNKR ligand expression profile that is modulated by HIV-1 infection.
367 We compiled a comprehensive panel of ligands to aNKR that included ULBP-1, ULBP-
368 2/5/6, ULBP-3. MIC-A, MIC-B, CD48, CD80, CD86, CD112, CD155, ICAM-1, ICAM-2, the
369 still largely unidentified ligands to NKp30, NKp44, and NKp46, and HLA-E, which is a non-
370 classical MHC that previous work from our lab suggested is expressed on 721 cells. The recent
371 identification of HLA-F as the ligand to the activating KIR3DS1 receptor on NK cells also
372 prompted us to extend our analyses to include this ligand (8, 9). We chose to analyze the
373 expression of HLA-F directly with the use of an HLA-F specific antibody and indirectly with the
374 use of a KIR3DS1-Fc chimeric molecule. Two ligands to iNKRs, HLA-C and the HLA-A were
375 also included in our study. The reason for this was to corroborate the well-established
376 observation that HLA-A expression is modulated by HIV infection and the recent finding that
377 HLA-C expression on HIV-infected CD4+ T cells is influenced by the viral strain used to infect
378 them (10). Examining HLA-A expression in both K562 and 721 confirmed that our in-house cell
379 lines were indeed HLA-null as expected, corroborating previous work from our lab showing that
380 a pan-MHC-1 antibody failed to stain these cell lines.
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381 The work presented here offers a more comprehensive characterization of the aNKR
382 ligand profiles of 721 and K562, as the antibody panel used in this thesis research probes a
383 broader range of aNKR ligands than has been done previously. This work also marks the first
384 head to head comparison of both HLA null cell lines.
385 Upon HIV infection, two clear populations of infected cells can be observed among
386 iCD4: infected cells that maintain surface expression of CD4 (iCD4+) and infected cells where
387 the cell surface expression of CD4 has been downregulated by the HIV-associated proteins Nef
388 and Vpu (iCD4-) (11, 12). Early work examining HIV-mediated CD4 downregulation suggested
389 that infected cells with downregulated CD4 were at a later stage of infection (12). It is also
390 plausible that these cells were more productively infected. Indeed, experiments in this thesis will
391 show that these cells expressed higher levels of the p24 viral capsid protein, which serves as a
392 marker of infection. Despite these differences, a review of the existing literature demonstrated a
393 lack of consensus in the way iCD4 should be examined. Some work analyzed iCD4- exclusively,
394 while the remainder disregarded the differences between both infected subsets entirely and
395 combined them to analyze total iCD4. As such, we were also interested in characterizing the
396 different aNKR ligand expression profiles of HIV-1-infected CD4+ T cells that maintained or
397 downregulated cell surface CD4. In so doing, we aimed to add to our current understanding of
398 how these two HIV-1-infected T cell subsets differed and further emphasize that they should be
399 considered separately.
400 Considering this, the ultimate goal of my thesis research was to compile a thorough
401 repertoire of the aNKR ligands expressed on the cell types commonly used to stimulate NK cells
402 in in vitro and ex vivo activation models, including K562, 721, and HIV-1-infected CD4+ T cells.
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403 Literature Review
404 1.3: History of the HIV pandemic
405 The first cases of acquired immunodeficiency syndrome (AIDS) were observed in the
406 early 1980s and shortly after, in 1983, the human immunodeficiency virus subtype 1 (HIV) was
407 identified as its causative agent (13, 14). Since its discovery, HIV has continued to disseminate
408 globally and is responsible for what is now one of the deadliest viral pandemics in history. As of
409 2015, almost 37 million people globally are living with HIV, while 35 million have died of
410 AIDS-related illness since the beginning of the epidemic (15). Therapeutic advances allowing for
411 the administration of highly-active antiretroviral therapy (HAART) with increasing potency and
412 tolerability and reduced toxicity have been instrumental in limiting the spread of the virus.
413 However, attempts at designing an effective HIV vaccine have not been so successful. Despite
414 the continually increasing availability of highly effective treatment, HIV continues to target
415 vulnerable high-risk populations and 2015 saw an additional 2 million people become newly
416 infected (15). In Canada, the prevalence of HIV continues to increase and is disproportionately
417 represented in men who have sex with men (53% of all Canadians living with HIV), injection
418 drug users (19% of all Canadians living with HIV), and First Nations, Métis, and Inuit peoples
419 (9% of all Canadians living with HIV) (16-18). It is evident that treatment alone is insufficient to
420 control HIV transmission and that these methods need to be supplemented with new approaches
421 arising from continued preventative and curative research.
422 1.3.2: Viral Structure
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423 HIV is a lentivirus of the Retroviridae family. The icosahedral HIV virion is enveloped
424 by a host cell-derived lipid bilayer that contains viral glycoproteins and some cellular membrane
425 proteins acquired from the host cell membrane (Figure 1). The virion contains a capsid-enclosed
426 genome composed of two copies of linear, negative-sense, single-stranded RNA (ssRNA)
427 molecules. The HIV genome has 3 major coding regions: gag, which encodes structural proteins
428 like the capsid protein p24, pol, which encodes the viral protease (PR), reverse transcriptase
429 (RT), and integrase (IN), and env, which encodes viral glycoproteins (Figure 1). The HIV
430 genome also contains several additional accessory genes, for a total of 9 open reading frames
431 within a genome of less than 10 kilobases (19, 20). In addition to the viral genome, the capsid
432 contains the viral nucleocapsid (NC), IN, RT, and the accessory protein Vpr.
433 As with most retroviruses, HIV is characterized by significant genetic heterogeneity and
434 these variants comprise three major phylogenetic groups: M (main), O (outlier), and N (non-M or
435 -O) (21-23). Group M variants make up the majority of global infection and can be further
436 subdivided into nine clades (A-K), of which clades A, B, and C are most prevalent (24).
437 Furthermore, there are two different strains of HIV, HIV-1 and HIV-2, which have similar
438 structures and genome, but are associated with different pathologies. HIV-2, largely confined to
439 West Africa, is less pathogenic and less transmissible than the pandemic strain HIV-1 (25).
- 7 -
440 Figure 1
441 Figure 1: Schematic diagram of the HIV virion and genome. The enveloped HIV virion contains an encapsidated 442 genome the codes for Gag, Pol, and Env, in additional to other accessory proteins (see text).
443 Image Source: adapted from Karlsson 2008 (26) and Peterlin 2003 (27).
444 1.3.3: Kinetics of infection
445 The majority of HIV infections arise as a result of sexual transmission through the genital
446 tract or rectal mucosa (28, 29). In vitro experiments using mucosal tissue and studies in non-
447 human primate models have helped elaborate how HIV infection is established. In this context,
448 HIV infection begins when free virions or cell-associated virus crosses the mucosal barrier into
449 the lamina propria (30-32). Within the lamina propria, HIV preferentially targets resting and
450 activated CD4+ T cells, but will also target macrophages and dendritic cells (DCs) (32-34). These
451 small populations of infected cells will propagate locally and produce virus until their numbers
452 are sufficient to disseminate into draining lymph nodes and other lymphoid tissues, establishing a
453 systemic infection (30-32). At this point, latently infected viral reservoirs have been established
454 and will sustain long-term self-propagating infection.
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455 At the cellular level, the mature HIV virion will attach to target CD4 T cells through
456 interactions between the viral envelope (Env) proteins gp41 and gp120 and the amino-terminal
457 immunoglobulin (Ig) domain of CD4 (35). Binding to CD4 is insufficient for infection, which
458 also requires interactions with the additional cell surface proteins CCR5 and CXCR4 (Reviewed
459 in 36). Conformational changes in gp120 that are induced by CD4 binding permit its interaction
460 with these co-receptors (37, 38). Subsequently, gp41 will also undergo a conformational change
461 that results in the fusion of the viral and host cell membranes and release of the viral capsid into
462 the host cell (38).
463 Membrane fusion is followed by uncoating events that are incompletely understood and
464 by the RT-catalyzed reverse transcription of the ssRNA HIV genome into linear double-stranded
465 DNA (dsDNA) (35, 39). RT lacks proofreading ability and frequently introduces mutations into
466 the viral genome (40, 41). The low fidelity of RT in conjunction with the rapid in vivo viral
467 turnover drive HIV’s extensive genetic variation and ability to rapidly adapt to host- and drug-
468 mediated selection pressures (42, 43). RT-dependent DNA synthesis also requires viral NC,
469 which is a nucleic acid chaperone, and may be facilitated by the accessory protein Viral
470 Infectivity Factor (Vif) (44, 45). Newly synthesized viral DNA is transported to the nucleus as
471 part of a preintegration complex (PIC) that also includes IN, RT, matrix (MA) and Viral Protein
472 R (Vpr) proteins (46). Vpr targets the PIC to the nucleus through interactions with the cellular
473 nuclear import machinery and can also arrest infected cells in the G2 phase of the cell cycle (47,
474 48). The transported viral DNA can then be integrated into the host genome by IN. These steps
475 make up the early phase of HIV’s viral life cycle. During the late phase, infected cells can either
476 enter the productive cycle whereby new viruses are assembled and released to propagate
- 9 -
477 infection or the integrated viral DNA can remain transcriptionally silent. Infected cells
478 containing transcriptionally silent viral DNA form viral reservoirs that can persist in HAART-
479 treated individuals and that can be reactivated following treatment interruption, a major
480 impediment to achieving a sterilizing cure for this disease (49-51).
481 The late phase of the productive HIV replication cycle begins with the synthesis of viral
482 mRNA transcripts that are translocated back to the cytoplasm, where they are translated. The
483 viral proteins Transactivator of Transcription (Tat), Regulator of Expression of Virion protein
484 (Rev), and Negative Regulatory Factor (Nef) are synthesized first and facilitate the transcription
485 and nuclear export of unspliced or singly-spliced mRNAs encoding Gag, Gag-Pol, Env, Viral
486 Protein Unique (Vpu), Vif, and Vpr (35). Tat is a transcriptional activator that binds to the Tat-
487 responsive element on nascent RNA transcripts and stimulates transcription elongation (35, 52,
488 53). Rev binds to Rev-response elements on nascent unspliced mRNA and shuttles it out of the
489 nucleus, despite unspliced mRNAs normally being retained in the nucleus (35, 54, 55). Once in
490 the cytosol, HIV RNA will dimerize and two copies of full length viral RNA will be packaged
491 into new viral particles (35). Together with the Gag-Pol precursor, Gag and HIV RNA will
492 assemble into an immature viral particle at the plasma membrane (35, 56). The viral RNA
493 genome will be packaged into the forming viral particle and encapsidated. The newly formed
494 viral particle will bud off the infected host cell.
495 Following budding, PR cleaves Gag and Gag-Pol to produce MA, capsid (CA), and NC,
496 which will rearrange during maturation to form the infectious viral particle (35, 56). Efficient
497 budding relies on the function of the accessory proteins Vpu and Nef. The viral Env protein
498 gp160 (precursor to gp120) and host CD4 are both produced in the endoplasmic reticulum (ER)
- 10 -
499 and premature gp160-CD4 interactions can impair the transport of gp160 to the plasma
500 membrane and the formation of viral particles (57). To prevent this, Vpu targets CD4 for
501 degradation, thus removing it from the ER (58-60). Similarly, Nef downregulates cell surface
502 CD4, which prevents premature binding of Env to CD4 and reinfection of an infected cell by its
503 own budding virions (61).
504 Figure 2
505 Figure 2: HIV replication cycle. (1) Viral attachment and fusion require interactions between envelope 506 glycoproteins CD4, as well as the co-receptors CCR5 and CXCR4. (2) The uncoated ssRNA genome is reverse 507 transcribed into dsDNA by the viral reverse transcriptase. (3) Newly synthesized DNA is translocated to the as part 508 of the preintegration complex and integrated into the host genome. (4) Viral mRNA is transcribed in the nucleus and 509 transported to the cytoplasm. (5) Viral proteins are translated and the replicated viral genome is packaged into 510 nascent viral particles at the cell membrane. (6) Viral particles bud off from the cell, coating themselves in host 511 membrane, and will continue to mature into infectious virions. See text for more details.
512 Image Source: Adapted from Deeks 2015 (62)
513 1.3.4: HIV immunopathogenesis
514 HIV infection follows a well-defined time course that begins with primary infection.
515 Within 3-6 weeks following infection, between 50-70% of individuals will experience acute
516 retroviral syndrome (ARS) (19, 63). Although the clinical manifestations of ARS and their - 11 -
517 severity vary greatly between individuals, common symptoms include fever, fatigue and general
518 malaise, pharyngitis, maculopapular rash, and lymphadenopathy (64, 65). While ARS usually
519 resolves within two weeks, lymphadenopathy and malaise may persist (19, 66). During primary
520 infection, plasma viral loads increase dramatically and peak around 3 weeks following infection
521 (29, 65, 67). Increases in viral load are accompanied by reductions in the levels of peripheral
522 blood CD4+ T cells that are rarely completely reversed without treatment (68). Early T cell
523 depletion is particularly significant in the gut, where mucosal T cells serve as the initial target of
524 infection following vaginal or rectal transmission. As these T cells are responsible for
525 maintaining gut homeostasis, their loss results in enteropathy, bacterial translocation, and
526 inflammation, which recruits more T cells to the site of damage (Reviewed in 69). This cycle of
527 infection and inflammation continues to provide HIV with new targets to infect, permitting rapid
528 viral expansion and establishing a chronic state of immune activation. Unsurprisingly, primary
529 infection is associated with innate immune activation, characterized by an intense cytokine
530 response and activation of NK and NKT cells (70-72). HIV-specific cytotoxic lymphocyte (CTL)
531 responses can also be observed, with the first CD8+ T cell responses arising during peak viremia
532 and peaking 1-2 weeks later, as viremia declines (29, 73, 74). Anti-HIV antibody responses arise
533 more slowly, within approximately 3 months of infection and remain present for years following
534 infection (19, 74-76).
535 The initial viral load spike and the immune responses it induces are followed by a long
536 period of asymptomatic infection, during which time symptoms are mild, if present, and viral
537 load decreases to a set point. However, viral replication continues during this time and peripheral
538 CD4+ T cell numbers steadily decline (77-80). Although the latent stage can last decades, in the
- 12 -
539 absence of treatment, the majority of infected individuals will progress to AIDS with a median
540 time from infection of 10 years. The onset of AIDS is characterized by a drop in CD4+ T cell
541 numbers to below 500 cells/μL (19). This potent immune suppression predisposes infected
542 individuals to opportunistic infections that often prove fatal. Paradoxically, chronic immune
543 activation can also contribute to disease progression and render HIV-infected individuals more
544 vulnerable to inflammatory diseases (81, 82). HAART administration can halt disease
545 progression and reverse CD4+ T cell depletion, however, this treatment cannot eliminate non-
546 replicating viral reservoir cells and must be administered for life to prevent viral rebound. As
547 such, much of the research directed towards prevention has focused on modulating immune
548 responses that occur early in infection and precede the establishment of viral reservoirs,
549 highlighting the importance of innate immune responses.
550 1.3.5: Discovery and function of NK cells
551 NK cells are innate immune lymphocytes that were first identified by their ability to
552 spontaneously kill allogeneic tumor cells in vitro (83, 84). NK cells develop in the bone marrow
553 from the same common lymphoid progenitor that generates mature B and T cells (Reviewed in
554 85). As with all innate immune mediators, NK cells can lyse target cell without prior antigen
555 sensitization and their responses occur much quicker than adaptive immune responses, which
556 earned them the title of ‘natural killers’ (86, 87). The mechanism through which NK cells
557 targeted tumor cells was addressed by the ‘missing-self’ hypothesis, which theorizes that NK
558 cells can recognize self-proteins – specifically, MHC class I molecules – and are induced to kill
559 targets cells where surface expression of these proteins is downregulated (Reviewed in 88). It has
560 subsequently been confirmed that MHC-I molecules are downregulated in tumor cells and that
- 13 -
561 this results in NK cell activation upon interaction (89). MHC-I molecules are also downregulated
562 during viral infections and times of cellular stress. Correspondingly, NK cells are now thought of
563 as the first line of defense against virally-infected and transformed cells.
564 The primary way in which NK cell kill is through the contact-dependent release of
565 cytotoxic granules by a process called degranulation. Upon interaction with a target cell that
566 results in NK cell activation, an immunological synapse will form at the point of contact and
567 secretory lysosomes containing granzyme and perforin mobilize towards it (90-92). Following
568 granule exocytosis, perforin forms pores in the target cell that allow granzyme to enter and
569 cleave its targets, ultimately resulting in target cell death (Reviewed in 93). The rapid, non-
570 specific contact-dependent lysis of target cells is an important innate immune function, but NK
571 cell can also collaborate with adaptive immunity. Through their FcγRIIIA (CD16) cell surface
572 receptors that bind to the Fc portion of human IgG, NK cells can detect antibody-coated target
573 cells and mediated antibody-dependent cellular cytotoxicity (ADCC) (94). CD16
574 heterodimerizes with FcϵRIγ chains or CD3-ζ chains, whose cytoplasmic tails contain immune
575 tyrosine-based activating motifs (ITAM) (95). Ligation of CD16 triggers the phosphorylation of
576 these ITAM motifs and, subsequently, NK cell cytotoxicity (96). Finally, NK cells are also an
577 important cellular source of interferon-γ (IFN-γ) and can also produce and secrete several other
578 chemokines and cytokines (2, 97). Through the production of these signaling molecules and
579 direct interactions, NK cells can recruit other cell types to sites of inflammation and infection
580 and shape T and B cell responses (98-100). Because of their functional plasticity, NK cells serve
581 as a unique bridge between innate and adaptive immunity.
582 1.3.6: NK cells in HIV
- 14 -
583 NK responses are an important component of immunity against many viruses, including
584 HIV. Certain combinations of killer Ig-like receptors (KIR) on NK cells and their human MHC-I
585 or HLA ligands have been associated with protection against HIV infection and slower disease
586 progression (101-104). The HIV-derived peptide motifs presented by those HLA molecules may
587 also contribute to protection, as HLA/KIR interactions with specific viral peptides modulates NK
588 cell responses in vitro (105). Much like tumor cells, the cell surface expression of MHC-I
589 molecules is reduced on HIV-infected cells. Mediated by the viral accessory protein Nef, HLA
590 downregulation protects infected cells from CTL responses, but makes them targets for NK cell
591 missing-self responses (106, 107). However, HIV has evolved to evade such responses by
592 selectively downregulating HLA molecules. HLA-C and -E expression is maintained on the
593 surface of infected cells, preserving interactions with inhibitory NK cell receptors (iNKRs) and
594 abrogating NK cell anti-HIV responses (108, 109).
595 Additionally, secondary analyses of HIV vaccine trials have highlighted the NK cell
596 mediated-ADCC as an important correlate of vaccine-induced protection (7). ADCC activity has
597 also been associated with improved viral control in infected individuals (110, 111). More
598 recently, it has been proposed that NK cells are capable of memory-like responses and that
599 memory NK cells maintain prolonged anti-viral responses in a simian immunodeficiency virus
600 (SIV) model. Specifically, NK cells from macaques infected with SIV or a chimeric SIV with an
601 HIV envelope were observed to be capable of antigen-specific lysis of target cells that had been
602 pulsed with HIV protein components (112). Furthermore, NK cells from animals vaccinated
603 against adenovirus 26-vectored SIV elicited vaccine antigen-specific responses 5 years after
604 vaccination (112). While the existence of memory NK cells has not yet been confirmed in
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605 humans, human cytomegalovirus (CMV) infection induces the preferential expansion of
606 CD94/NKG2C-expressing NK cells, which can mediate memory-like responses against CMV
607 (113-115).
608 1.4: NK cell licensing and activation
609 Anti-viral NK cell responses and NK cell functions as a whole are governed by the
610 balance of signals transmitted through aNKR and iNKR. In this context, NK cells can be
611 activated one of two ways. First, upon encountering target cells that do not express ligands to
612 iNKR, inhibitory signaling through these receptors will be abrogated, allowing for activation in
613 its absence. Secondly, NK cells interacting with target cells that do express ligands to aNKR will
614 experience an increase in activating signaling through those receptors. Physiologically, however,
615 most cells express ligands to both iNKR and aNKR and whether an NK cell will be activated
616 depends on the number and strength of each signal type. Additionally, NK cells can also be
617 activated by cytokines produced by other immune cells during infection and inflammation. For
618 example, interleukin (IL)-2 can promote NK cell survival, proliferation, and IFN-γ secretion,
619 while IL-12 and IL-18 act synergistically to stimulate NK cell IFN-γ production via promotion of
620 JAK/STAT signaling (116-122). Several other cytokines capable of triggering NK cell
621 cytotoxicity have been identified, but the mechanisms through which they activate NK cells have
622 not been conclusively defined.
623 The strength of mature NK cell responses is also influenced by a process known as
624 education, whereby HLA-KIR interactions during NK cell development dictate tolerance to self
625 and cytotoxicity against non-autologous and virally-infected or transformed cells (123). The
- 16 -
626 importance of HLA-KIR interactions in directing NK cell functional competency was first
627 suggested by the observation that NK cells developing in MHC-devoid environments lacked the
628 characteristic ability to lyse MHC-null cells (124, 125). KIRs comprise an important subset of
629 NKRs and recognize subsets of HLA molecules on the surface of putative target cells (126, 127).
630 The inhibitory KIRs (iKIRs) that govern NK cell education have immunoreceptor tyrosine-based
631 inhibition motifs (ITIMs) in their long cytoplasmic tails and are categorized by their number of
632 extracellular Ig-like domains (2D or 3D) (127). NK cells lacking iKIRs for self-HLA molecules
633 are hyporesponsive following in vitro stimulation and fail to lyse MHC-deficient bone marrow
634 transplants and mutations in KIR ITIMs confer hyporesponsiveness, even when those KIRs are
635 in the presence of their cognate ligands (123, 128, 129). Three different models for NK cell
636 education have been proposed. The first ‘arming’ model hypothesizes that NK cells are
637 constitutively unresponsive and uneducated and that iKIR engagement by their HLA ligands
638 during development confers functional competence (123). In contrast, a second ‘disarming’
639 model postulates that NK cells are constitutively and chronically responsive and will become
640 anergic in the absence of iKIR/HLA interactions (130). A third ‘rheostat’ model proposes that
641 education tunes NK cell responsiveness according to the number of inhibitory self-HLA
642 receptors on NK cells and the affinity of each receptor for their cognate ligand (131, 132).
643 According to this model, NK cells expressing two or more iKIRs would be more functional than
644 NK cells expressing only one. This functional enhancement could improve NK cell licensing
645 (arming model) or oppose chronic activation and anergy (disarming model).
646 1.4.2: Models for the study of NK cell activation
- 17 -
647 HLA-null cells are commonly used to study NK cell activation. HLA-null cells lack the
648 surface expression and, on occasion, the mRNA transcripts of the MHC-I molecules HLA-A, -B,
649 and -C. NK cell that interact with HLA-null cells will experience abrogation of inhibitory
650 signaling through the iKIRs that bind HLA-A, -B, and -C antigens. HLA-null cells do, however,
651 express ligands to aNKRs and activating signaling through these receptors, in tandem with the
652 suppression of inhibition through iKIRs, will activate NK cells and stimulate missing-self
653 responses. The two cell lines utilized by our lab for these purposes are the K562 and 721.221
654 (721) cell lines. The K562 cell line was first derived from a patient with chronic myelogenous
655 leukemia (CML) in blast crisis (133, 134). Phenotypically, K562 resemble non-adherent myeloid
656 progenitor cells and can spontaneously develop erythrocyte-, granulocyte-, and monocyte-like
657 characteristics (135). The karyotype of this cell line appears to vary according to its time in
658 culture, increasing from hypodiploid to near triploid in longer cultures (136-138). 721 is a B cell
659 lymphoblastoid cell line that was transformed and immortalized by Epstein-Barr virus (EBV)
660 infection and thus expresses the EBV genome (139-141). 721 are characterized by the complete
661 absence of class I HLA mRNA transcripts and α-chains as a result of gamma ray-induced
662 mutation in the HLA complex, but this cell line does express endogenous non-classical HLA-E
663 (139, 142, 143). While both cell lines lack HLA-A, -B, and -C antigens, we observed that they
664 stimulated NK cells differently and this likely reflects differences in the expression of ligands to
665 aNKR (4).
666 NK cells are activated during the course of viral infections and autologous HIV-infected
667 CD4+ T cells (iCD4) can also be used to activate NK cells in vitro. As has been mentioned,
668 downregulation of HLA-A and -B on the surface of iCD4, although this is impaired somewhat by
- 18 -
669 maintained expression of HLA-C, is partially responsible for activating NK cells and triggering
670 missing-self responses. Furthermore, HIV infection can also modulate the expression of ligands
671 to aNKRs and upregulation of those ligands on iCD4 likely also contributes to their ability to
672 activate NK cells (144-146). Following their contact-dependent activation, NK cells inhibit
673 further viral replication via the production of chemokines (5, 147). Activated NK cells can
674 secrete CC-chemokines CCL3, CCL4, and CCL5, which compete with HIV for binding of the
675 CCR5 co-receptor on CD4+ T cells and block viral entry (148, 149). However, NK cell-mediated
676 inhibition of HIV replication is incompletely understood and other NK cell functions may
677 contribute to this process.
678 1.5: Characterization of activating NK cell receptors (aNKRs) and their ligands
679 1.5.2: NKG2 receptors
680 One of the major aNKR is C-type lectin-like receptor family natural-killer group 2
681 member D (NKG2D), which is expressed on all NK cells and CD8+ T cells (150-152). This type
682 II transmembrane protein is encoded by a gene in the NKG2 complex on human chromosome 12
683 and contains no known signaling motif in its intracellular domain (153-155). Rather, this
684 receptor homodimerizes with the adaptor protein DNAX-activating protein of 10 kDa (DAP10)
685 for signal transduction and stability (152, 154, 156). Upon activation of the NKG2D and DAP10
686 homodimer, DAP10, which possesses a YXXM tyrosine-based motif, will recruit
687 phosphoinositide kinase-3 (PI3K) and growth factor receptor-bound protein 2 (Grb2) (152, 157).
688 In humans, NKG2D exclusively interacts with DAP10, however, a shorter isoform of NKG2D in
- 19 -
689 mice is also capable of binding to DNAX-activating protein of 12 kDa (DAP12) (158, 159). The
690 signaling pathways of NKG2D and other NKG2 receptors are illustrated in Figure 3.
691 Figure 3
692 Figure 3: NKG2 receptor signaling. Ligation of NKG2D and CD94/NKG2C, which associate with the adaptors 693 DAP10 and DAP12, respectively, results in NK cell activation. Interaction between HLA-E and CD94/NKG2A 694 results in inhibition of NK cell responses (see text).
695 Image source: Iwaszko 2012 (160)
696 NKG2D is notable in that several distinct MHC class-I-like ligands can bind to this single
697 invariant receptor. In humans, NKG2D recognizes members of the retinoic acid early transcript 1
698 (RAET1) or UL16-binding protein family (ULBP1-6; named for their ability to bind the UL16
699 glycoprotein of CMV) and the MHC class-I-chain-related proteins (MIC) A and B (161-165).
700 ULBP1-3 are glycosylphosphatidylinositol (GPI)-linked glycoproteins, while ULBP-4 and -5 are
701 transmembrane glycoproteins (163, 166). Like classical MHC-I molecules, MIC-A and -B are
702 highly polymorphic and have a structure that includes three external domains (α1-3), a
703 transmembrane domain, and a cytoplasmic domain (165, 167). Similarly, the Ig-like domains of
704 the ULBPs are comparable to the α1 and α2 domains of MHC-I molecules (168). The structure
705 of the NKG2D ligands is illustrated in Figure 4. However, ULBP and MIC proteins distinguish
- 20 -
706 themselves from their MHC-I counterparts in their inability to bind β2-microglobulin and
707 associate with transporter-associated proteins, making these proteins unable to bind peptide and
708 present antigen (169-172). Both ULBP and MIC proteins are classified as stress ligands, as they
709 are generally not expressed on healthy cells, but are upregulated by cellular stressors such as
710 viral and bacterial infection and cellular transformation (173-180). Engagement of NKG2D by
711 its cognate ligands on target cells triggers NK cell cytotoxicity through the regulation of NK cell
712 granule polarization and degranulation by NKG2D/DAP10 signaling (181). Furthermore,
713 signaling through NKG2D is potent enough to trigger NK cell activation despite concurrent
714 inhibitory self-recognition signaling (162, 182).
715 Figure 4
716 Figure 4: NKG2D ligands. The two families of NKG2D ligands, MIC and ULBP, have characteristic structures (see 717 text). TM+CYT = transmembrane and cytoplasmic domains. GPI = glycosyl-phosphatidyl-inositol anchor.
718 Image source: Fernández-Messina 2012 (183)
719 In addition to NKG2D, other members of the C-type lectin-like NKG2 family can
720 contribute to NK cell activation, including NKG2C, NKG2E, and NKG2H. These three NKG2
721 receptors bind to the non-classical MHC class Ib HLA-E (184, 185). Binding of HLA-E to a set
722 of nonamer peptides derived from the leader sequences of HLA class I molecules or HLA-G
723 allows for the stable expression of this ligand on the cell surface of a broad distribution of tissues
- 21 -
724 (186, 187). Through the binding of these leader peptides, HLA-E also functions to indirectly
725 monitor the expression of HLA class I molecules and can also be used by NK cells to monitor
726 cellular integrity through its binding to virally-, bacterially-, and stress-derived peptides (188-
727 191).
728 NKG2C, NKG2E, and NKG2H form heterodimers with the invariant CD94 glycoprotein,
729 which is disulfide-linked to an NKG2 glycoprotein (192, 193). NKG2C also noncovalently
730 associates with DAP12, whose presence is required for efficient cell surface expression of
731 CD94/NKG2C (194-196). NK cell activation upon ligation of DAP12 results in the
732 phosphorylation of its single cytoplasmic immunoreceptor tyrosine base activating motif (ITAM)
733 and ensuing recruitment of the tyrosine kinases spleen tyrosine kinase (Syk) and zeta-chain-
734 associated protein kinase 70 (ZAP-70) (194, 196, 197). NKG2C expression is associated with
735 terminal differentiation of NK cells and improved cytotoxicity (198). NKG2C-expressing NK
736 cells may be particularly important in the context of CMV infection, where this subset of NK
737 cells expands, enhances IFN-γ production, and acquires a mature phenotype (113, 115, 199,
738 200). Furthermore, NKG2C deletions have been associated with increased risk for HIV infection
739 and faster progression to AIDS and elevated frequencies of NKG2C+ NK cells are also observed
740 during HIV infection (114, 201, 202). However, considering that coincident HIV and CMV
741 infections are common and the interconnectedness of HIV progression and CMV reactivation, it
742 is difficult to conclude whether these findings are due to HIV infection alone or underlying CMV
743 infection (203-205). Whereas NKG2C is expressed on the surface of most NK cells, recent work
744 has shown that human NKG2E is retained in the cell cytoplasm where it forms intracellular
745 complexes with DAP12 and CD94 (206). In contrast, NKG2H, a splice variant of NKG2E, is
- 22 -
746 transported to the cell membrane in conjunction with CD94 and DAP12 (207). The contribution
747 of NKG2E and NKG2H signaling to NK-mediated anti-viral responses is currently unknown.
748 CD94/NKG2C and NKG2E share extensive homology with the inhibitory
749 CD94/NKG2A, to which HLA-E also binds and with a greater affinity (208, 209). The
750 cytoplasmic domain of NKG2A contains an ITIM that is phosphorylated upon engagement by
751 HLA-E and subsequently recruits Src homology region 2 domain-containing phosphatase (SHP)-
752 1 and SHP-2 to inhibit NK cell functions (210). Furthermore, co-stimulating NKG2A and
753 NKG2C when they are co-expressed on NK cells results in net inhibition (211). As such, it is
754 plausible that NKG2A signaling regulates NK cell stimulation by other activating NKG2 to limit
755 potential autoreactivity against self-HLA-E.
756 1.5.3: Natural cytotoxicity receptors (NCRs)
757 NCRs comprise the other major family of aNKRs and include the type I transmembrane
758 proteins NKp30, NKp44, and NKp46 (212-214). NCRs were first identified by their shared
759 ability to stimulate NK cell-mediated tumor cell lysis in vitro, although they share limited amino
760 acid or structural homology (214-217). Some similarities include the possession of one or two
761 extracellular ligand binding Ig-like domains and a transmembrane domain that can interact with
762 ITAM-containing signaling adaptor molecules (summarized in Figure 5).
763 NKp30 is encoded by the NKP30 gene, which is located in the class III region of the
764 human MHC locus on chromosome 6, and is expressed on all mature NK cells (214). NKp30 has
765 one extracellular domain that can recognize the tumor cell ligand B7-H6, the tegument protein
766 pp65 of human CMV, and the viral hemagglutinin (HA) of the orthopox family members
- 23 -
767 vaccinia virus and ectromelia virus (218-221). NKp30 can also bind heparin sulfate
768 proteoglycans on the surface of target cells, although the exact identity of its cellular ligand is
769 unknown (222-224). The molecule has an intracellular domain that lacks any signaling domain
770 and instead activates NK cells through the CD3ζ homodimers and CD3ζ/FcRγ heterodimers that
771 interact with the positively charged arginine residue in its transmembrane domain (218, 225).
772 FcRγ and CD3ζ have one and three intracellular ITAMs, respectively, which are phosphorylated
773 upon ligand binding and recruit Syk and ZAP-70 (226, 227). Ligation of NKp30 stimulates NK
774 cell cytotoxicity, degranulation, and IFN-γ production (228, 229). Interestingly, NKp30 has an
775 immunosuppressive splice variant known as NKp30c and signaling through this variant impairs
776 NK cell degranulation and TNF-α secretion, while stimulating IL-10 secretion (229).
777 NKp44 is also encoded in the class III region of the human MHC locus and possesses a
778 single V-type extracellular ligand-binding domain (217, 230-232). The transmembrane domain
779 of NKp44 contains a positively charged lysine residue and associates with and signals through
780 dimerized DAP12 (224, 233). NKp44 is unique among NCRs as it possesses an ITIM within its
781 cytoplasmic tail, although this ITIM is believed to be non-functional (231, 234). As with NKp30,
782 NKp44 has been shown to bind to heparin sulfate proteoglycans and a variety of tumor, viral,
783 and bacterial ligands (223, 235-238). Recently, proliferating cell nuclear antigen and NKp44L,
784 which shares sequence similarity with the mixed-lineage leukemia protein 5, have been
785 identified as cellular ligands for NKp44 (239, 240). However, these ligands are only expressed
786 intra-cellularly and cell surface ligands to NKp44 have yet to be identified (239, 241, 242).
787 NKp46 was the first NCR identified and is encoded by the NKP46 gene on chromosome
788 19 (213, 224). NKp46 is expressed on all NK cells, regardless of their activation state, and is also
- 24 -
789 expressed by some subsets of T cells and innate lymphoid cells (ILCs) (243-245). NKp44 has
790 two extracellular C2-type Ig domains that are connected by a hinge region, a single
791 transmembrane domain, and a cytoplasmic domain that lacks any signaling motifs (224, 246,
792 247). As with NKp30, the arginine residue in NKp46’s transmembrane domain interacts with the
793 FcRγ and CD3ζ adaptor proteins, which provide the signaling required for NK cell activation
794 (224). Additionally, homodimerization of NKp46, which would bring the transmembrane and
795 intracellular domains into close proximity, appears to be required for NK cell activation (248).
796 NKp46 can bind to the HA of influenza and certain poxviruses, as well as the hemagglutinin
797 neuraminidase of the Sendai virus and Newcastle disease virus (219, 220, 249, 250). NKp46 can
798 also bind to vimentin, a cellular ligand expressed on mycobacterium tuberculosis-infected cells,
799 targeting vimentin-expressing cells for NK cell lysis (251). As with all the NCRs, NKp46 can
800 bind to heparin sulfate proteoglycans, but more work will be required to determine which
801 specific cellular ligands that NKp46 and the other NCRs can engage (222, 223).
802 Figure 5
803 Figure 5: Structure of natural cytotoxicity receptors. Natural cytotoxicity receptors contain extracellular ligand- 804 binding domains and transmembrane domains that can interact with different adaptor molecules (see text).
805 Image source: Kruse 2014 (224)
- 25 -
806 1.5.4: Additional activating receptors
807 2B4
808 2B4, a member of the signaling lymphocyte activation molecule (SLAM) family of
809 receptors, is an NK receptor that can activate NK cells directly or provide co-stimulatory signals
810 that enhance NCR-, NKG2D-, and DNAM-1-mediated NK cell activation (252, 253). 2B4 is
811 expressed by all NK cells and some T cell subsets and binds to CD48, which is itself a SLAM
812 family receptor (254, 255). CD48 is broadly expressed on lymphoid and myeloid-derived cells,
813 where several viral infections can modulate its expression (144, 256-258). In humans,
814 stimulation of 2B4-expressing NK cells with CD48-expressing target cells induces NK cell
815 activation, cytotoxicity, and IFN-γ secretion (255, 259, 260). Upon ligation, 2B4 can also
816 transmit inhibitory signals and is thought to collaborate with MHC class I receptors to limit
817 autoreactive NK cell responses (254). The complexity of 2B4 signaling is due in part to the four
818 immunoreceptor tyrosine-based switch motifs (ITSM) in its intracellular domain. Following 2B4
819 engagement, 2B4 will be recruited into lipid rafts and its ITSM motifs will be phosphorylated
820 and recruit SLAM-associated protein (SAP) (261, 262). SAP inhibits the interactions between
821 2B4’s fourth ITSM and the negative regulatory molecules SHP-1, SHP-2, SH2 domain-
822 containing inositol 5'-phosphatase (SHIP), and C-terminal Src kinase (CSK), suppressing
823 inhibitory signaling (261, 263). Correspondingly, NK cells from patients with X-linked
824 lymphoproliferative disease – who lack a functional SAP protein – are inhibited upon 2B4
825 ligation (264-266). It has thus been proposed that the duality of 2B4 signaling is regulated by the
826 availability of SAP in the NK cell cytoplasm (267). The density of CD48 on target cells and
827 expression levels of 2B4, which is internalized and downregulated following ligation, are also
- 26 -
828 thought to influence the balance of inhibitory and activating 2B4 functions (267, 268).
829 Furthermore, 2B4 association with lipid rafts is essential for ITSM phosphorylation and 2B4-
830 mediated NK cell activation. Co-engagement of 2B4 and iNKR, such as KIRs and
831 CD94/NKG2A, interferes with lipid raft recruitment and phosphorylation of 2B4 and provides
832 another level of regulation (262, 269).
833 DNAX accessory molecule-1 (DNAM-1)
834 The leukocyte adhesion DNAM-1 (also known as CD226) is a transmembrane
835 glycoprotein, part of the Ig superfamily, and an aNKR (270). This receptor is encoded on
836 chromosome 18 and features two extracellular Ig-like domains and a cytoplasmic tail with three
837 tyrosine residues (271). DNAM-1 is expressed on monocytes, DCs, T cells, and virtually all
838 resting NK cells, where its engagement results in in Fyn-mediated phosphorylation of the
839 tyrosine residues and induction of NK cell cytotoxicity (270, 271). DNAM-1 can bind to both the
840 δ and α isoforms of CD112 (also known as Nectin-2), which differ from each other in their
841 transmembrane and cytoplasmic regions, and the nectin-like family member CD155 (also known
842 as the poliovirus receptor). However, binding to CD155 appears to predominate DNAM-1’s
843 interactions with its cognate ligands (271). The expression of both cellular ligands for DNAM-1
844 can be upregulated by cellular stress and are modulated by pathological conditions, including
845 cancer and infectious disease (272, 273). Interactions between DNAM-1 and its ligands regulate
846 NK cell adhesion and the induction phase of activation and cytolytic activity and contribute to
847 tumor immune surveillance (270, 271, 274, 275). DNAM-1 signaling is also a promoter of NK
848 cell cytokine secretion during inflammation, the induction of which can be reduced by
849 competition with other receptors for CD155 (276). The expression of CD112 and CD155 on DCs
- 27 -
850 has also been shown to be an important regulator of a selection process known as DC editing,
851 whereby NK cells can kill immature DCs after interacting with them (277-279). Interactions
852 between DNAM-1 and CD155, which is upregulated on T cells following activation of the T cell
853 receptor, may also be involved the NK cell-mediated elimination of activated T cells (280).
854 DNAM-1 signaling has also been implicated in the differentiation and expansion of the still
855 controversial memory NK cells during murine CMV infection and loss of DNAM-1 is associated
856 with impaired viral control and prolonged virus clearance (281, 282). Furthermore, cell surface
857 expression of CD112 and CD155 can be modulated by several viral proteins, impairing anti-viral
858 NK cell responses, and facilitating immune evasion (283-285).
859 Lymphocyte function-associated antigen-1 (LFA-1)
860 Interestingly, efficient DNAM-1 function requires the co-expression of the αLβ2 integrin
861 LFA-1, which is expressed on all NK cells (286). In NK cells isolated from patients who lack
862 LFA-1, DNAM- signaling is defective, but can be restored through genetic reconstitution of
863 LFA-1 expression (286). LFA-1 binds to intercellular adhesion molecule (ICAM)-1 and ICAM-
864 2, interactions that are critical for forming the immunological synapse and tight adhesion of NK
865 and target cells (287-292). Engagement of LFA-1 by ICAM-1 and -2 can also induce the
866 polarization of cytolytic granules in NK cells, which is a crucial early step in triggering contact-
867 dependent cytolysis (293-295). LFA-1-mediated NK cell activation occurs downstream of the
868 rapid phosphorylation of vav guanine nucleotide exchange factor-1 (Vav1) and the kinase protein
869 tyrosine kinase 2-β (Pyk2) and reorganization of the actin cytoskeleton (296-298). LFA-1
870 signaling and the ability of NK cells to form synapses with target cells requires the stable
- 28 -
871 tethering of ICAMs to α-actinin and to the ezrin/radixin/moesin (ERM) protein ezrin in the actin
872 cytoskeleton (289, 299-302).
873 CD28
874 Similarly, interaction with target cells expressing the B7 molecules CD80 and CD86 can
875 also trigger granule polarization and cytotoxicity in NK cells (303-306). The contribution of
876 CD80 and CD86 to T cell co-stimulation through their interactions with CD28 and cytotoxic T-
877 lymphocyte-associated protein 4 (CTLA-4) is well established (Reviewed in 307). Nevertheless,
878 it was believed that the majority of human peripheral blood NK cells (pNKs) did not express
879 CD28 and that interaction of CD80 and CD86 with NK cells would be independent of this
880 receptor (308). Furthermore, murine studies reported that the NK-mediated cytotoxicity induced
881 by stimulation with CD80- and CD86-expressing target cells could not be abrogated by blocking
882 CTLA-4 or CD28 (305, 306). However, human NK cells do express a variant of the T cell CD28
883 receptor, although expression levels are generally low and vary among individuals (303, 309).
884 Moreover, blocking of CD28 on human pNKs was able to completely eliminate NK cell-
885 mediated killing of target cells that expressed CD80 and CD86 (303). Despite these findings, the
886 nature of the mechanisms that underlie CD80- and CD86-induced NK cell activation remain
887 incompletely understood.
888 KIR3DS1
889 Although ITIM-containing KIRs are perhaps the most important family of iNKRs, a
890 smaller number contain ITAM motifs and can transmit activating signals upon ligation. One such
891 aNKR is encoded by the conserved allelic variant KIR3DS1, which shares its locus with alleles
- 29 -
892 encoding inhibitory KIR3DL1 allotypes (310). As an activating KIR, KIR3DS1 (3DS1) has a
893 characteristic short ITAM-containing cytoplasmic tail and transmembrane domain that associates
894 with DAP12 (310, 311). Signaling through 3DS1 induces NK cell cytotoxicity and IFN-γ
895 secretion (311). Carriage of 3DS1 has been associated with the acquisition risk and disease
896 outcome of a number of viral infections and most prominently with delayed disease progression
897 in HIV infected patients (101, 312-314). The Bernard laboratory reported that carriage of 3DS1
898 in its homozygous form was associated with protection from HIV infection in HIV exposed
899 seronegative subjects (104). Despite the extensive shared homology of the extra-cellular domains
900 of 3DS1 and KIR3DL1 (3DL1), while 3DL1 has been conclusively shown to bind to a subset of
901 HLA-A and -B molecules that contain a Bw4 motifs, attempts at identifying ligands to 3DS1
902 have been less successful (310, 315-317). Recently, however, the non-classical MHC class I
903 molecule HLA-F was identified as a high affinity ligand to 3DS1 (8, 9). Specifically, the open
904 conformer form of HLA-F, in which HLA class I heavy chains are not bound to β2m or peptide,
905 binds to 3DS1 on NK cells and triggers degranulation and production of pro-inflammatory
906 cytokines (8, 318). The cell surface expression of HLA-F is tightly regulated (319). HLA-F is
907 expressed at the surface of some tumors and B cell lines, but rarely in normal cells, where it is
908 thought to be retained in the endoplasmic reticulum (320-323). Although HLA-F is not expressed
909 on the surface of resting lymphocytes, its expression can be induced by activation (324).
910 Furthermore, HIV infection reduces the expression of HLA-F on previously activated T cells,
911 particularly on late stage infected CD4 T cells (8). Together these findings demonstrate that
912 HLA-F expression can be modulated during the course of inflammatory responses and infection.
- 30 -
913
914
915 Previous work from our lab that assessed the number of NK cells and NK cell functional
916 subsets activated by the two HLA-null cell lines K562 and 721.221 (721) found that K562 and 721
917 differed significantly in their capacities as NK cell stimuli. Specifically, we observed that 721
918 activated a larger fraction of NK cells and induced significantly greater numbers of cytokine and
919 chemokine secreting NK cells, as measured by IFN-γ and CCL4 production. We also observed
920 that, while K562 induced lower frequencies of all functional subsets relative to 721, they
921 preferentially induced degranulation, which was measured by CD107α expression. HLA-null cells
922 do not express the MHC-I human leukocyte antigens (HLA)-A, -B, and -C, which are ligands to
923 the inhibitory killer immunoglobulin-like receptors on NK cells, but do express ligands to
924 activating NK cell receptors. We were interested in studying whether, K562 and 721 had different
925 expression profiles of ligands to activating NK cell receptors and whether this might explain their
926 different abilities to stimulate NK cells.
927
928
- 31 -
929 Chapter 2: Manuscript 1 - The HLA-null cell lines K562 and 721.221 express different
930 ligand profiles for activating NK cell receptors.
931
932
933
934
935
936
937
938
939
940
941
942
- 32 -
943 The HLA-null cell lines K562 and 721.221 express different ligand profiles for activating NK 944 cell receptors. 945 946 947 948 949 Alexandra Tremblay-McLean1,2, Julie Bruneau3,4, Bertrand Lebouché1,5,6, and Nicole F. 950 Bernard1,2,6,7 951 952 953 1Research Institute of the McGill University Health Center, Montréal, Québec, Canada 954 2Division of Experimental Medicine, McGill University, Montréal, Québec, Canada 955 3Department of Family Medicine, Université de Montréal, Montréal, Québec, Canada 956 4Centre de Recherche de Centre Hospitalier de l’Université de Montréal, Montréal, Québec, 957 Canada 958 5Department of Family Medicine, McGill University, Montréal, Québec, Canada 959 6Chronic Viral Illness Service, McGill University Health Centre, Montréal, Québec, Canada 960 7Division of Clinical Immunology, McGill University Health Centre, Montréal, Québec, Canada 961 962 963 964 965 966 967 Mailing address: Dr. Nicole F. Bernard, Research Institute of the McGill University Health 968 Centre, Glen Site, 1001 Décarie Boulevard, Block E, Rm E03.3380, Montréal, Québec, Canada, 969 H4A 3J1. 970 Tel: (514) 934-1934 ext:42697 971 E-mail: [email protected] 972 973 974 Funding: This study received support from the Canadian Institutes for HIV Research grant #
975 MOP-142494. A. Tremblay-McLean was supported by Canadian Association for HIV Research
976 Abbvie Master’s Award in Basic Science.
977 978 Word Count: 4212
- 33 -
979 2.1: Author contributions and acknowledgments
980
981 ATM and NFB were responsible for study design, data analysis, and manuscript preparation. JB
982 and BL provided subject samples.
983
984 We would like to acknowledge the generosity of Dr. Galit Alter, who provided our 721 cell line,
985 and the assistance of several lab members. In particular, Xiaoyan Ni and Tsoarello Mabanga for
986 helping to maintain the HLA-null cell lines and Drs. Irene Lisovsky and Gamze Isitman for
987 helping with the development of the multi-parametric flow cytometry panels.
988
989
990
991
992
993
- 34 -
994 2.2: Abstract
995 Natural Killer (NK) cells mediate innate immune responses to stressed, virally-infected,
996 and transformed cells. NK cell activation depends on the integration of signals received through
997 inhibitory (iNKR) and activating NK receptors (aNKR). HLA-null cells can be used in vitro to
998 stimulate and study NK cell activation. Previous work by our group comparing NK cell
999 stimulation by the HLA-null cell lines K562 and 721.221 (721) found that they stimulated
1000 different frequencies of NK cell functional subsets. As both these cell lines lack surface
1001 expression of classical MHC-1 antigens, a subset of which are ligands to iNKR, differences in
1002 NK function following K562 and 721 stimulation may reflect differences in the expression of
1003 ligands for aNKR. Using multiparametric flow cytometry we analyzed the expression of the
1004 following aNKR ligands on these HLA-null cell lines: ULBP-1, ULBP-2/5/6, ULBP-3, MIC-A,
1005 MIC-B, CD48, CD80, CD86, CD112, CD155, ICAM-1, ICAM-2, HLA-F, HLA-E, and the
1006 ligands to NKp30, NKp44, NKp46, and KIR3DS1. Expression was measured as the frequency of
1007 cells expressing each ligand and as an expression score, which reflected the frequency of positive
1008 cells multiplied by the mean fluorescence intensity of staining for each ligand. We found that,
1009 both cell lines expressed ICAM-1, ICAM-2, and HLA-F. K562 exclusively expressed ULBP-
1010 2/5/6, ULBP-3, CD112, CD155, and the ligand to NKp30, while 721 expressed characteristically
1011 high levels of MIC-B, CD48, CD80, CD86, HLA-E, and the ligand to NKp44. The ligands
1012 expressed on 721 can engage a larger number of aNKR on NK cells, which may, in part, explain
1013 their ability to activate a larger fraction of these cells and elicit different functions, compared to
1014 K562.
- 35 -
1015 2.3: Introduction
1016 Natural Killer (NK) cells are a subset of lymphocytes that direct innate immune
1017 responses to kill stressed, virally-infected, and transformed cells (Reviewed in 1). NK cells can
1018 interact with target cells and lyse them directly via the release of cytotoxic granules containing
1019 perforin and granzyme (2, 3). Activated NK cells can also secrete a broad range of cytokines and
1020 chemokines that can activate adaptive immune cells to lyse target cells, bridging the innate and
1021 adaptive immune systems (4-6). Regardless of the method of lysis, an NK cell must first be
1022 activated to elicit a response. The activation state of an NK cell results from the integration of
1023 different signals transmitted through its activating (aNKR) and inhibitory (iNKR) receptors (6,
1024 7). Activation can result from the loss of inhibitory signaling, when there are no ligands for
1025 iNKR to engage, in conjunction with sustained aNKR signaling, or from the engagement of
1026 aNKR by their ligands that overwhelms inhibitory signaling through iNKR (1, 6, 8, 9). However,
1027 in vivo, NK cells interacting with target cells will receive a variety of signals through both
1028 receptor types and whether this results in activation or not depends on the number and strength of
1029 each type of signal transmitted.
1030 A common method to activate NK cells is by co-culturing them with HLA-null cells.
1031 The most frequently used HLA-null cells include the myelogenous leukemia K562 and B-
1032 lymphoblastoid 721.221 (721) cells lines, which do not express the major histocompatibility
1033 complex class I (MHC-I) human leukocyte antigens (HLA) -A, -B, or -C antigens on their
1034 surface (10-12). As these cells are incapable of engaging the inhibitory killer immunoglobulin-
1035 like receptors (KIR) on NK cells, which recognize subsets of HLA-A, -B, and -C, inhibitory
1036 signaling through these receptors is abrogated (1, 6). As signals from these inhibitory receptors
- 36 -
1037 oppose those from aNKR, their removal allows the engagement of aNKR by their ligands to
1038 activate NK cells (13).
1039 Previous work from our lab demonstrated that the K562 and 721 HLA-null cell lines
1040 stimulated NK cells differentially to secrete the cytokine IFN-γ, the chemokine CCL4, and to
1041 express the degranulation marker CD107a. Specifically, we observed that 721 activated a greater
1042 fraction of NK cells than did K562 and that stimulation with 721 preferentially induced IFN-γ
1043 and CCL4 secretion, while K562 more potently stimulated degranulation (14). In the absence of
1044 the expression of the major ligands for iNKR on the surface of K562 and 721, it is likely that
1045 ligands to aNKR regulate NK cell activation and differences in their expression profiles might
1046 explain the different capacities of these two cell lines to activate NK cells. Indeed, the expression
1047 of some ligands to aNKR was shown to differ on both cell lines, although these differences
1048 remain incompletely characterized (15-20).
1049 There are two major classes of aNKR. The first is the C-type lectin NKG2D receptor that
1050 binds to a family of MHC-I like molecules expressed on healthy cells only after periods of
1051 cellular stress (15, 21). These include the human cytomegalovirus UL-16 binding proteins
1052 (ULBP) and MHC-I related chain (MIC) proteins (17, 22, 23). The second includes the natural
1053 cytotoxicity receptors (NCR) NKp30, NKp44, and NKp46, which can bind to membrane-
1054 associated heparan sulfate glycosaminoglycans and viral hemagglutinin (24-31). Despite these
1055 findings, the cellular ligands to NCRs remain incompletely identified.
1056 In addition to the two major aNKR families, signaling through several other receptors can
1057 contribute to NK cell activation. These include CD244/2B4 and the NK-T-B cell antigen (NTB-
1058 A), which are CD2 family receptors that engage CD48 to trigger NK cell cytotoxicity (32).
- 37 -
1059 Another receptor, expressed on virtually all NK cells, is the leukocyte adhesion molecule DNAX
1060 accessory molecule-1 (DNAM-1) that binds to CD112 (Nectin-2) and CD155 (poliovirus
1061 receptor) ligands (16, 33, 34). Signaling through DNAM-1 can stimulate NK cells when it is co-
1062 expressed with the lymphocyte function-associated antigen 1 (LFA-1). LFA-1 can bind the
1063 integrins ICAM-1 and ICAM-2 on target cells, bridging NK and target cells and forming the
1064 immunological synapse (35-39). Additionally, target cells expressing the T cell co-stimulatory
1065 B7 molecules CD80 and CD86 can stimulate NK cells (40-42). The nature of this signaling is
1066 still poorly understood, but it has been suggested that NK cell activation by CD80 and CD86
1067 depends on a CD28 variant expressed on NK cells (41, 43).
1068 In contrast to HLA-A, -B, and -C, which signal through iNKRs, the non-classical HLA-E
1069 and F can contribute to NK cell activation through the engagement of aNKRs. Much like its
1070 classical MHC-I counterparts, HLA-E interacts with the inhibitory C-type lectin-like receptor
1071 NKG2A, which is heterodimerized with CD94, and with the aNKRs NKG2E and -C (44-47).
1072 HLA-F is a more recent addition to the family of ligands to aNKR and has been shown to
1073 stimulate degranulation and cytokine production through its binding to KIR3DS1 on NK cells
1074 (48, 49).
1075 To determine whether the differences observed in frequency and function of NK cell
1076 subsets stimulated by K562 and 721 are related to differences in their expression profiles of
1077 ligands to aNKRs, we analyzed the expression of a comprehensive panel of aNKR ligands on
1078 both HLA-null cell lines by multi-parametric flow cytometry. As ligands to inhibitory KIR are
1079 not expressed on HLA-null cell lines, it is likely that expression of ligands to aNKRs plays a role
1080 in modulating differential NK cell responses to these cells. This research sought to better
- 38 -
1081 characterize the phenotype of two NK cell stimuli, the HLA-null cell lines K562 and 721, in
1082 order to better understand the mechanisms governing their ability to activate NK cells.
- 39 -
1083 2.4: Methods
1084 2.4.1: Origin and preparation of K562 and 721 cell lines
1085 K562 cells (American Type Culture Collection, Manassas, VA) and 721 cells (a kind gift
1086 from Dr. Galit Alter; Harvard University) were cryopreserved in in 10% dimethyl sulfoxide
1087 (DMSO; Sigma-Aldrich, St. Louis, MO) with 90% fetal bovine serum (FBS; Wisent, Inc. St.
1088 Bruno, QC, Canada) were thawed and cultured in RPMI 1640 medium supplemented with 10%
1089 FBS; 2mM L-glutamine; 100IU/mL penicillin; 100mg/mL streptomycin (R10, all from Wisent)
1090 for at least one full passage before staining. 721 and K562 that were cultured in R10
1091 supplemented with 400IU/mL of recombinant human IL-2 (rhIL-2; Chiron Corp., Emeryville,
1092 CA) for 3 days served as positive controls.
1093 2.4.2: Antibody staining and acquisition
1094 Both HLA-null cell lines were stained within two passages of thawing and in triplicate
1095 with UV Live/Dead ® Fixable Blue cell stain kit, as per manufacturer’s directions
1096 (Thermofisher, Waltham, MA) and then were stained with the following antibody-fluorochrome
1097 conjugates (monoclonal antibody clones) specific for the following cell surface antigens, which
1098 were organized into 4 panels for the purpose of analysis: (Panel 1) ULBP-1-PerCP (170818),
1099 ULBP-2/5/6-APC (165903), ULBP-3-PE (166510), MIC-A-APC (159227), MIC-B-AlexaFluor
1100 700 (236511; all from R&D Systems, Minneapolis, MN), (Panel 2) CD48-PE (BJ40), CD80-
1101 BV421 (2D10), CD86-PE-Dazzle594 (IT2.2; all from BioLegend, San Diego, CA), CD112-
1102 AlexaFluor 700 (610603; R&D), CD155-PE-Cy7 (SKII.4), (Panel 3) ICAM-1-Pacific Blue
1103 (HCD54), and ICAM-2-PE (CBR-1C2/2; all from BioLegend). Cells were also stained with
- 40 -
1104 recombinant human IgG1 Fc chimeras NKp30-Fc, NKp44-Fc, and NKp46-Fc (all from R&D) as
1105 a part of Panel 3. A final HLA Panel comprised the conjugated antibody HLA-E-PE-Cy7 (3D12;
1106 BioLegend), as well as an unconjugated recombinant human IgG1 Fc chimera KIR3DS1-Fc
1107 (R&D) and 3D11, an unconjugated primary antibody against HLA-F (a kind gift from Dr. Daniel
1108 Geraghty, Fred Hutchison Research Institute, Seattle, WA). Briefly, cells were resuspended in a
1109 96-well V bottom plate (Sarstedt, Nümbrecht, Germany) at a concentration of 1x106 cells per
1110 100μL of Dulbecco’s phosphate buffered saline (dPBS; Wisent) and stained with the Live/Dead
1111 reagent. TruStain FcX reagent (BioLegend) was used to minimize non-specific Fc receptor
1112 interactions and cells were stained with conjugated antibodies from Panel 1, 2, 3, and HLA for
1113 30 min in the dark at room temperature (RT). For staining with any of the recombinant human
1114 IgG1 Fc chimeras or 3D11, cells were prepared as previously described, with the exception that
1115 they were stained with protein chimeras or primary antibody on ice for 40 min. After washing,
1116 binding of Fc chimera proteins was detected using a polyclonal anti-human IgG (Fc gamma-
1117 specific)-PE secondary antibody, while 3D11 binding was detected using a polyclonal F(ab’)2
1118 anti-mouse IgG-APC secondary antibody (both from eBioscience) for 20 min on ice. Following
1119 staining, all cells were washed and fixed with 2% paraformaldehyde (PFA; Santa Cruz, Dallas
1120 TX). Between 400,000 and 600,000 events were acquired on an LSRFortessa x20 within 24hrs
1121 (BD). Unstained, single stained controls (CompBead; BD), fluorescence minus one, and
1122 secondary antibody alone controls were used for multi-colour compensation and gating purposes.
1123 Flow cytometric analysis was performed using FlowJo software version 10 (TreeStar, Ashland
1124 OR). All the antibodies used and their manufacturers are listed in Appendix A.
1125 Results are presented as the frequency of cells expressing each aNKR ligand and the
1126 expression score for each of these aNKR ligands. The expression score for a ligand was
- 41 -
1127 calculated by multiplying the frequency and mean fluorescence intensity (MFI) of cells
1128 expressing that ligand. As K562 and 721 have different levels of autofluorescence, the MFI was
1129 first background corrected against the MFI of unstained cells.
1130 2.4.3: Statistical analysis
1131 Statistical analysis was performed using GraphPad Prism version 6 (GraphPad Software,
1132 Inc., La Jolla, CA). Mann-Whitney tests were used to determine the significance of differences in
1133 the frequency or expression score of a cell surface ligand between 2 unpaired groups. Data on the
1134 frequency and expression score of ligand expression is represented as median (range), with those
1135 three values representing each of the triplicate values. P-values less than 0.05 were considered
1136 significant.
- 42 -
1137 2.5: Results
1138 2.5.1: Comparison of the frequencies of K562 and 721 expressing ligands to aNKR
1139 We hypothesized that differences in aNKR ligands expressed by K562 and 721 could
1140 explain their different abilities to stimulate NK cells. We used flow cytometry to determine the
1141 frequency of K562 and 721 cells expressing ligands to aNKR. Figure 1 shows the percentage of
1142 cells expressing ligands detected by antibody panels 1, 2, 3, and HLA. As IL-2 can upregulate
1143 the surface expression of these ligands, HLA-null cells cultured for 3 days in rhIL-2-containing
1144 media served as positive controls for the comparison of ligand expression on unstimulated cells.
1145 For Panel 1 (Fig. 1A), we observed that a median of 64.9(63.1, 66.7)%, 21.3(19.5, 28.4)%, and
1146 24.3(24.2, 28.1)% of K562 cells expressed ULBP-2/5/6, ULBP-3, and MIC-B, respectively. Less
1147 than 10% of these cells expressed the other ligands in panel 1. On the other hand, less than 10%
1148 of 721 expressed the stress ligands in this panel except for MIC-B, which was present on
1149 76.9(66.2, 77.9)% of these cells. Thus, a higher frequency of K562 than 721 cells expressed
1150 ULBP-2/5/6 and ULBP-3, while a lower frequency of K562 than 721 expressed MIC-B. As
1151 shown in Figure 1B, 15.4(11.3, 21.1)%, 88.6(88.2, 88.7)%, 0.4(0.3, 0.5)%, 67.5(66.5, 68.6)%
1152 and 99.1(99.0, 99.2)% of K562 expressed CD48, CD80, CD86, CD112, and CD155,
1153 respectively. Virtually all 721 cells were positive for CD48, CD80, and CD86, while 3.8(3.4,
1154 3.8)% and 6.8(6.0, 9.9)% expressed CD112 and CD155. Therefore, CD48 and CD86 was present
1155 on a lower frequency of K562 than 721, while a higher frequency of K562 than 721 cells
1156 expressed CD112 and CD155.
1157 All K562 and 721 cells expressed ICAM-1 and ICAM-2 (Fig. 1C). We found that
1158 73.0(71.4, 74.5)% and 24.7(20.1, 40.5)% of K562 expressed the ligand to NKp30 and NKp44,
- 43 -
1159 whereas 0.8(0.4, 1.0)% and 41.7(39.5, 42.8)% of 721 expressed these ligands. As such,
1160 compared to 721, a higher frequency of K562 expressed the ligand to NKp30, while a lower
1161 frequency expressed the ligand to NKp44. Neither cell line expressed the ligand to NKp46.
1162 HLA-E was present on 21.1(12.9, 21.7)% of K562 and 42.7(40.4, 48.5)% of 721 cells (Fig. 1D).
1163 HLA-F was detected on 55.6(50.9, 59.0)% and 60.2(47.6, 60.3)% of K562 cells and 80.9(75.1,
1164 85.4)% and 66.8(60.5, 69.6)% of 721 cells stained with 3D11 and the KIR3DS1-Fc chimera
1165 protein, respectively. Thus, both K562 and 721 expressed HLA-E and HLA-F, though there was
1166 a trend towards the frequency of expression of these markers being lower on the former cell line.
1167 In sum, we observed that variable frequencies of K562 cells expressed ULBP-2/5/6, ULBP-3,
1168 MIC-B, CD48, CD80, CD112, CD155, ICAM-1, ICAM-2, the ligands to NKp30 and NKp44,
1169 HLA-E, and HLA-F. In contrast, 721 expressed MIC-B, CD48, CD80, CD86, ICAM-1, ICAM-
1170 2, the ligand to NKp44, HLA-E, and HLA-F. K562, but not 721, expressed ULBP-2/5/6, ULBP-
1171 3, CD112, CD155, and the ligand to NKp30. 721, but not K562, expressed CD86 and a higher
1172 frequency of 721 than K562 cells expressed MIC-B, CD48, CD80, and HLA-E.
1173 2.5.2: Comparison of the expression score for ligands to aNKR on the K562 and 721 HLA-
1174 null cell lines
1175 In addition to determining the frequency with which each HLA null cell line expressed
1176 the aNKR ligands studied, we also examined the intensity with which these ligands were
1177 expressed by measuring the MFI of staining by the antibodies specific for each ligand. We then
1178 multiplied the frequency of cells positive for each ligand by the MFI of ligand staining to obtain
1179 a fluorescence expression score.
- 44 -
1180 The expression score for panel 1 ligands had a pattern similar to that observed for the
1181 frequency of these ligands on K562 and 721 cells. As shown in Figure 2A, the expression score
1182 for ULBP-2/5/6 was 252(240, 258) on K562, compared to 0(0, 0) on 721. Additionally,
1183 compared to 721, ULBP-3 and MIC-B expression was 10.8-fold greater and 2.4-fold lower on
1184 K562 cells. For panel 2 ligands, K562 had expression scores for CD48, CD80, and CD86 that
1185 were almost 300-fold, 2.4-fold, and almost 800-fold lower than those of 721 (Fig. 2B).
1186 Meanwhile, the expression scores of CD112 and CD155 were 65-fold and 122-fold greater on
1187 K562 than on 721. Both cell lines had similar expression scores for ICAM-1, ICAM-2, and the
1188 ligand to NKp46 (Fig. 2C). However, K562 had an expression score for the ligand to NKp30 of
1189 216(203, 234), while 721 did not express this ligand, and expressed the ligand to NKp44 at
1190 levels 2.3-fold less than those observed on 721. Of the HLA ligands studied, only HLA-E was
1191 expressed differentially on K562 and 721, with K562 having an expression score for this ligand
1192 that was 2-fold lower than that of 721 (Fig. 2D. Overall, these findings mirror the trends
1193 observed in frequency of aNKR ligand expression. Namely, that K562 exclusively express the
1194 aNKR ligands ULBP-2/5/6, ULBP-3, CD112, CD155, and the ligand to NKp30, whereas 721
1195 express MIC-B, CD48, CD80, CD86, the ligand to NKp44, and HLA-E at levels greater than
1196 those observed on K562.
- 45 -
1197 2.6: Discussion
1198 A subset of the MHC-I HLA-A, -B, and -C antigens bind to inhibitory KIRs on NK cells.
1199 In the absence of any cell-surface iNKR ligands, differential expression of ligands to aNKR on
1200 HLA-null cell lines plays a more important role in regulating their ability to interact with and
1201 activate NK cells. Previous work from our lab found that the two HLA-null cell lines K562 and
1202 721 activated different frequencies of NK cells exhibiting any permutation of IFN-γ and/or
1203 CCL4 secretion or CD107α expression, as well as several of the functional subsets defined by
1204 these activities (14). A complete characterization of the ligands to aNKR expressed on different
1205 NK cell stimuli is crucial to predict their stimulatory activity and to understanding the
1206 mechanisms that underlie that activation. Here, we performed phenotypic analyses to examine
1207 the expression profiles of ligands to aNKRs on K562 and 721. We observed that while both cell
1208 lines shared expression of some of these ligands, they each possessed a characteristic expression
1209 profile. Although prior studies have partially characterized the expression profiles of ligands to
1210 aNKRs on K562 and 721, in this study, we included a larger and more comprehensive panel of
1211 ligands and, for the first time, compared the ligand profiles on these HLA-null cells at the same
1212 tie and in the same experiments.
1213 We observed that both HLA-null cell lines expressed the adhesion molecules ICAM-1
1214 and ICAM-2 and HLA-F, the ligand for the activating KIR3DS1 receptor on NK cells. This
1215 discovery was unexpected, as recent work that identified HLA-F as the ligand for KIR3DS1 did
1216 not find that K562 expressed HLA-F (48, 49). K562 cells exclusively expressed ULBP-2/5/6,
1217 ULBP-3, CD112, CD155, and the ligand for the activating NCR NKp30. These findings are
1218 consistent with previous reports that ULBP ligands are preferentially expressed on K562 and
- 46 -
1219 with the observation that K562 express the tumor ligand B7-H6, which can bind NKp30 (19, 50).
1220 The stress ligands ULBP-2/5/6 and ULBP-3 bind to the C-type lectin NKG2D and CD112 and
1221 CD155 bind to DNAM-1 (16, 22, 23, 34). As such, K562 has a ligand profile that is consistent
1222 with being able to bind to and activate NK cells through NKG2D, DNAM-1 and NKp30, while
1223 721 are incapable of engaging these aNKRs. NKG2D signaling has been shown to play a crucial
1224 role in NK cell cytotoxic granule polarization, degranulation, and cytotoxicity (51). The ability
1225 of K562 to induce signaling through this pathway may explain why this cell line preferentially
1226 stimulates NK cell degranulation, rather than cytokine or chemokine secretion (14).
1227 Unfortunately, no antibody currently exists that can discriminate between ULBP-2, -5, and -6
1228 and we are incapable of determining which combinations of these ligands are expressed on
1229 K562. However, it is possible that K562 express all three of these ligands, in addition to ULBP-
1230 3, and could, therefore, induce more potent signaling through NKG2D than 721, which express a
1231 single NKG2D ligand (MIC-B). Moreover, expression of NKp30 on NK cells has been
1232 correlated with both perforin expression and degranulation and engagement of this aNKR by
1233 K562 may also have contributed to their induction of degranulation (52).
1234 In contrast, 721 exclusively expressed CD48 and CD86 and expressed characteristically
1235 high levels of MIC-B, CD80, the ligand for NKp44, and HLA-E. Our findings confirm that
1236 HLA-E, the ligand to the aNKRs NKG2E and NKG2C and iNKR NKG2A on NK cells, is
1237 expressed by 721 cells (53). Stable cell-surface expression of HLA-E usually requires binding to
1238 a set of nonamer peptides derived from the leader sequences of HLA class I molecules or HLA-
1239 G, which are absent in the HLA-null 721 (54, 55). However, the HLA-E peptidome was recently
1240 shown to be less restricted that previously thought. HLA-E can also bind to an array of self-
1241 peptides in the absence of HLA class I signal peptides, permitting its stable expression and
- 47 -
1242 induction of NK cell cytotoxicity (56, 57). In addition, HLA-E is also capable of presenting
1243 Epstein-Barr virus (EBV)-derived peptides, like BZLF1, which would be present in the EBV
1244 transformed 721 cell line (58, 59). Yet, HLA-E/BZLF1 complexes are poorly recognized by NK
1245 cells and it is likely that HLA-E molecules presenting noncanonical self-make greater
1246 contributions to 721-induced NK cell activation (58).
1247 Engagement of the aNKR 2B4 by its ligand CD48 on 721 can induce low levels of IFN-γ
1248 production and concurrent signaling through 2B4 and other aNKRs can drive high levels of
1249 cytokine and chemokine production (6, 18). Although, the exact NKR that recognizes the co-
1250 stimulatory molecules CD80 and CD86 has not been identified, expression of these ligands on
1251 target cells can trigger NK cell-mediated cytolysis (41). Furthermore, signaling through NKp44,
1252 which is expressed on activated NK cells, induces the production of cytokines, like IFN-γ (25,
1253 60). Together, the ligands that are expressed on 721 are capable of engaging receptors that are
1254 important for the production of cytokines and chemokine, which may explain why 721 stimulate
1255 larger numbers of IFN-γ and CCL4 secreting NK cells (14). Additionally, the aNKR ligand
1256 expression profile of 721 is associated with binding to NK cells through a larger number of
1257 aNKR than K562. This may contribute to the observation that 721 activate greater numbers of
1258 NK cells, compared to K562 (14).
1259 For the purpose of this study, the ligands analyzed were included on the basis of their
1260 ability to stimulate NK cell responses through the engagement of aNKRs. However, it is
1261 important to consider that several of these ligands are capable of signaling through both aNKRs
1262 and iNKRs. CD112 and CD155, which signal through the activating DNAM-1, can also bind to
1263 the iNKR, T cell immunoreceptor with immunoglobulin and ITIM motifs (TIGIT) (61, 62).
- 48 -
1264 While both DNAM-1 and TIGIT are widely expressed on NK cells, the affinity of CD155 for
1265 TIGIT is greater than for DNAM-1 and TIGIT expression can reduce DNAM-1/CD155
1266 interactions in a dose-dependent manner (62-64). TIGIT has also been shown to compete with
1267 DNAM-1 for the binding of CD112. Furthermore, when transfected into the NK cell line YTS,
1268 TIGIT greatly limits NK-medicated cytotoxicity by disrupting cytotoxic granule polarization (64,
1269 65). Considering this, it is possible that CD112 and CD155, which are exclusively expressed on
1270 K562, contribute more to NK cell inhibition than activation and may be an additional reason why
1271 K562 activate a smaller fraction of NK cells, compared to 721 (14). Another aNKR ligand,
1272 HLA-E, similarly contributes to both NK cell activation and inhibition. HLA-E binds to the
1273 CD94/NKG2 family of NK cell receptors, which includes the activating NKG2E and -C and the
1274 inhibitory NKG2A and -B NKRs (44, 45). Interactions between NKG2A, which is expressed on
1275 the majority of NK cells, and HLA-E have been shown to predominate over interactions with
1276 NKG2C and surface expression of HLA-E is sufficient to save those cells from lysis by
1277 NKG2A+ NK cells (44, 45, 66). Despite this, work assessing NK cell stimulation by autologous
1278 HIV-infected CD4 T cells, which express HLA-E, found that carriage of NKG2A on NK cells
1279 was associated with improved activation (67). It is plausible that, while interactions between
1280 HLA-E on 721 and NKG2A on NK cells can impair NK cell activation, signaling through the
1281 other aNKR engaged by characteristic 721 ligands can compensate for this inhibitory input.
1282 In conclusion, we found that the two HLA-null cell lines K562 and 721 differed in their
1283 expression of ligands to activating NK cell receptors. Together, these experiments provide a
1284 more systematic assessment of the mechanisms underlying the stimulus properties of two cell
1285 types commonly used to study NK cell activation. The different aNKR ligand expression profiles
1286 of K562 and 721 are associated with the induction of different NK cell responses, however, more
- 49 -
1287 work that examines the specific contribution of each ligand-aNKR pair to different stages of NK
1288 cell activation will be required to confirm the functional effects of these phenotypic differences.
- 50 -
1289 2.7: Figure legends
1290 Figure 1: Different frequencies of K562 and 721 express ligands to activating NK cell
1291 receptors. Comparison of the frequency of K562 (white bars) and 721 (grey bars) expressing
1292 (A) Panel 1, (B) Panel 2, (C) Panel 3, and (D) HLA aNKR ligands. The frequency (%) of HLA-
1293 null cells expressing the indicated ligand is represented on the y-axis. Bar height and error bars
1294 represent the median and range for the data set. Each data point represents one of three replicates
1295 run concurrently.
1296 Figure 2: K562 and 721 differ in the expression score of their ligands to activating NK cell
1297 receptor. Comparison of the expression score of (A) Panel 1, (B) Panel 2, (C) Panel 3, and (D)
1298 HLA aNKR ligands on K562 (white bars) and 721 (grey bars). The expression score was
1299 calculated by multiplying the frequency and mean fluorescence intensity of the indicated ligand
1300 expression and is represented on the y-axis. Bar height and error bars represent the median and
1301 range for the data set. Each data point represents one of three replicates run concurrently.
- 51 -
1302 Figure 1: Different frequencies of K562 and 721 express ligands to activating NK cell
1303 receptors.
- 52 -
1304 Figure 2: K562 and 721 differ in the expression score of their ligands to activating NK cell
1305 receptor.
- 53 -
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55. Lee N, Goodlett DR, Ishitani A, Marquardt H, Geraghty DE. 1998. HLA-E surface expression depends on binding of TAP-dependent peptides derived from certain HLA class I signal sequences. J Immunol 160:4951-4960. 56. Kraemer T, Celik AA, Huyton T, Kunze-Schumacher H, Blasczyk R, Bade-Döding C. 2015. HLA-E: Presentation of a Broader Peptide Repertoire Impacts the Cellular Immune Response— Implications on HSCT Outcome. Stem Cells International 2015:346714. 57. Celik AA, Kraemer T, Huyton T, Blasczyk R, Bade-Döding C. 2016. The diversity of the HLA-E-restricted peptide repertoire explains the immunological impact of the Arg107Gly mismatch. Immunogenetics 68:29-41. 58. Jørgensen PB, Livbjerg AH, Hansen HJ, Petersen T, Höllsberg P. 2012. Epstein-Barr virus Peptide Presented by HLA-E is Predominantly Recognized by CD8(bright) Cells in multiple Sclerosis Patients. PLoS ONE 7:e46120. 59. Romagnani C, Pietra G, Falco M, Millo E, Mazzarino P, Biassoni R, Moretta A, Moretta L, Mingari MC. 2002. Identification of HLA-E-specific alloreactive T lymphocytes: a cell subset that undergoes preferential expansion in mixed lymphocyte culture and displays a broad cytolytic activity against allogeneic cells. Proc Natl Acad Sci U S A 99:11328-11333. 60. Castriconi R, Dondero A, Cantoni C, Della Chiesa M, Prato C, Nanni M, Fiorini M, Notarangelo L, Parolini S, Moretta L, Notarangelo L, Moretta A, Bottino C. 2007. Functional characterization of natural killer cells in type I leukocyte adhesion deficiency. Blood 109:4873-4881. 61. Stanietsky N, Simic H, Arapovic J, Toporik A, Levy O, Novik A, Levine Z, Beiman M, Dassa L, Achdout H, Stern-Ginossar N, Tsukerman P, Jonjic S, Mandelboim O. 2009. The interaction of TIGIT with PVR and PVRL2 inhibits human NK cell cytotoxicity. Proc Natl Acad Sci U S A 106:17858-17863. 62. Yu X, Harden K, C Gonzalez L, Francesco M, Chiang E, Irving B, Tom I, Ivelja S, Refino CJ, Clark H, Eaton D, Grogan JL. 2009. The surface protein TIGIT suppresses T cell activation by promoting the generation of mature immunoregulatory dendritic cells. Nat Immunol 10:48-57. 63. Wang F, Hou H, Wu S, Tang Q, Liu W, Huang M, Yin B, Huang J, Mao L, Lu Y, Sun Z. 2015. TIGIT expression levels on human NK cells correlate with functional heterogeneity among healthy individuals. European Journal of Immunology 45:2886-2897. 64. Stanietsky N, Rovis TL, Glasner A, Seidel E, Tsukerman P, Yamin R, Enk J, Jonjic S, Mandelboim O. 2013. Mouse TIGIT inhibits NK-cell cytotoxicity upon interaction with PVR. Eur J Immunol 43:2138-2150. 65. Liu S, Zhang H, Li M, Hu D, Li C, Ge B, Jin B, Fan Z. 2013. Recruitment of Grb2 and SHIP1 by the ITT-like motif of TIGIT suppresses granule polarization and cytotoxicity of NK cells. Cell Death Differ 20:456-464. 66. Valés-Gómez M, Reyburn HT, Erskine RA, López-Botet M, Strominger JL. 1999. Kinetics and peptide dependency of the binding of the inhibitory NK receptor CD94/NKG2-A and the activating receptor CD94/NKG2-C to HLA-E. The EMBO Journal 18:4250-4260. 67. Lisovsky I, Isitman G, Song R, DaFonseca S, Tremblay-McLean A, Lebouche B, Routy JP, Bruneau J, Bernard NF. 2015. A Higher Frequency of NKG2A+ than of NKG2A- NK Cells Responds to Autologous HIV-Infected CD4 Cells irrespective of Whether or Not They Coexpress KIR3DL1. J Virol 89:9909-9919.
57
1306
1307
1308
1309
1310 NK cells can be activated by autologous HIV-infected CD4+ T cells and will
1311 subsequently inhibit viral replication. It has been well established that the HIV viral accessory
1312 proteins Nef and Vpu can internalize the major histocompatibility complex (MHC)-1 molecules
1313 human leukocyte antigens (HLA)-A and -B, which are ligands to the inhibitory killer
1314 immunoglobulin-like receptors on NK cells. Similarly, the influence of HIV on inhibitory
1315 receptor expression on NK cells and the impact of these changes on NK-mediated anti-HIV
1316 responses has been well studied. In contrast, modulation of ligands to activating NK cell
1317 receptors by HIV infection and its contribution to anti-viral NK cell responses remains
1318 incompletely characterized. We wanted to address this knowledge gap by assessing the
1319 expression of a comprehensive panel of ligands to activating NK cell receptors on HIV-infected
1320 and -uninfected CD4+ T cells.
1321
1322
1323
1324 - 58 -
1325 Chapter 3: Manuscript 2 – Expression profiles of ligands for activating NK cell receptors
1326 on HIV infected and uninfected CD4+ T cells.
1327
1328
1329
1330
1331
1332
1333
1334
1335
1336
1337
1338
1339
1340 - 59 -
1341 Expression profiles of ligands for activating NK cell receptors on HIV infected and 1342 uninfected CD4+ T cells. 1343 1344 1345 1346 Alexandra Tremblay-McLean1,2, Julie Bruneau3,4, Bertrand Lebouché1,5,6, and Nicole F. 1347 Bernard1,2,6,7 1348 1349 1350 1Research Institute of the McGill University Health Center, Montréal, Québec, Canada 1351 2Division of Experimental Medicine, McGill University, Montréal, Québec, Canada 1352 3Department of Family Medicine, Université de Montréal, Montréal, Québec, Canada 1353 4Centre de Recherche de Centre Hospitalier de l’Université de Montréal, Montréal, Québec, 1354 Canada 1355 5Department of Family Medicine, McGill University, Montréal, Québec, Canada 1356 6Chronic Viral Illness Service, McGill University Health Centre, Montréal, Québec, Canada 1357 7Division of Clinical Immunology, McGill University Health Centre, Montréal, Québec, Canada 1358 1359 1360 1361 1362 1363 1364 Mailing address: Dr. Nicole F. Bernard, Research Institute of the McGill University Health 1365 Centre, Glen Site, 1001 Décarie Boulevard, Block E, Rm E03.3380, Montréal, Québec, Canada, 1366 H4A 3J1. 1367 Tel: (514) 934-1934 ext:42697 1368 E-mail: [email protected] 1369 1370 1371 Funding: This study received support from the Canadian Institutes for HIV Research grant #
1372 MOP-142494. A. Tremblay-McLean was supported by Canadian Association for HIV Research
1373 Abbvie Master’s Award in Basic Science.
1374 1375 Word Count: 5769 1376
- 60 -
1377 3.1: Author contributions and acknowledgements
1378
1379 ATM and NFB were responsible for study design, data analysis, and manuscript preparation. JB
1380 and BL provided subject samples.
1381
1382 We would like to acknowledge the assistance of several lab members. In particular, Ms. Xiaoyan
1383 Ni and Ms. Tsoarello Mabanga who KIR and HLA typed the study subjects. Dr. Irene Lisovsky
1384 and Dr. Gamze Isitman helped with the development of the multi-parametric flow cytometry
1385 panels. Dr. Irene Lisovsky and Dr. Rujun Song who provided the raw data for the analysis of
1386 inhibition of viral replication.
1387
1388
1389
1390
1391
1392
1393
- 61 -
1394 3.2: Abstract
1395 Natural Killer (NK) cells play a crucial role in the clearance of virally-infected cells, an
1396 ability that depends on the integration of signals received through inhibitory and activating NK
1397 receptors (iNKR and aNKR). HIV-infected CD4 T cells (iCD4) activate NK cells to inhibit HIV
1398 replication. HIV infection-dependent changes in the HLA ligands for iNKR on iCD4 are well
1399 documented. In contrast, less is known regarding the HIV infection related changes in ligands for
1400 aNKR on iCD4 T cells. Here, we compared the aNKR ligand profiles of early and late stage HIV
1401 iCD4s to uninfected CD4 (unCD4) T cells. We activated and infected CD4 T cells isolated from
1402 17 HIV-1 seronegative donors with HIVJR-CSF for 7 days. UnCD4 served as a control for the
1403 effect of HIV infection on aNKR. On day 7, the expression of ULBP-1, ULBP-2/5/6, ULBP-3,
1404 MIC-A, MIC-B, CD48, CD80, CD86, CD112, CD155, ICAM-1, ICAM-2, HLA-E, HLA-A2,
1405 HLA-C was analyzed by flow cytometry. Intracellular p24 staining was also used to distinguish
1406 iCD4 from uninfected CD4 cells present in the same culture (euCD4). We found that the iCD4
1407 that maintained CD4 surface expression (iCD4+) showed higher expression of aNKR ligands
1408 compared to unCD4, euCD4, and iCD4 where CD4 expression had been downregulated by HIV
1409 Nef. Meanwhile, the expression of aNKR ligands on iCD4- was similar to (ULBP-1, ULBP-
1410 2/5/6, ULBP-3, MIC-A, CD80, and ICAM-1) or significantly lower than (MIC-B, CD48,
1411 CD112, CD155, and ICAM-2) what was observed on unCD4 and euCD4. This suggests that HIV
1412 infection initially results in increased expression of aNKR ligands, but that this increase is
1413 transient and decreases to baseline or below concomitantly with the HIV-mediated
1414 downregulation of CD4 on the surface of infected cells. Additionally, the aNKR ligand profiles
1415 of unCD4 and euCD4 T cells did not differ, suggesting that exposure to virus of uninfected cells
1416 alone is insufficient for aNKR ligand modulation.
- 62 -
1417 3.3: Introduction
1418 Natural Killer (NK) cells are innate immune cells that mediate cytotoxic responses to
1419 stressed, virally-infected, and transformed cells (Reviewed in 1). NK cells can lyse target cells
1420 directly through the production of granzyme and perforin, or indirectly through antibody (Ab)-
1421 dependent mechanisms, through the production of cytokines and chemokines, and through
1422 activation of adaptive immune cells (2-4). The induction and potency of these responses depend
1423 on the activation state of the NK cell, which is regulated by signals transmitted through both
1424 activating and inhibitory NK cell receptors (aNKR and iNKRs) (4, 5). Signals from iNKR often
1425 dominate by interrupting signals from aNKR, the consequences of which explain tolerance to
1426 self (6). When signals from iNKR are interrupted NK cell activation can occur if target cells
1427 express ligands to aNKR (1, 4, 7, 8). However, in vivo most cells co-express variable
1428 combinations of ligands to both NKR types and NK cell activation will depend on the integration
1429 of the number and strength of all activating and inhibitory signals transmitted.
1430 NK cells express a variety of receptors. One of the major aNKRs is the C-type lectin
1431 NKG2D, which binds to a family of major histocompatibility complex (MHC)-I like molecules
1432 that includes the human cytomegalovirus UL-16 binding proteins (ULBP) and MHC-I related
1433 chain (MIC) proteins (9-11). The expression of these ligands is induced by cellular stress and
1434 they are otherwise not found on the surface of healthy cells (12). The other major family of
1435 aNKR comprises the natural cytotoxicity receptors (NCR) NKp30, NKp44, and NKp46 (9, 13-
1436 15). Although NCRs can bind membrane-associated heparan sulfate glycosaminoglycans and
1437 viral hemagglutinin, their cellular ligands remain incompletely identified (16-20).
- 63 -
1438 Several additional receptors that contribute to NK cell activation by target cells have been
1439 identified. Two members of the CD2 subfamily of receptors, CD244/2B4 and the NK-T-B cell
1440 antigen (NTB-A), can bind to CD48 and trigger NK cell cytotoxicity (21). The leukocyte
1441 adhesion molecule DNAX accessory molecule-1 (DNAM-1) is widely expressed on NK cells
1442 and engages both CD112 (Nectin-2) and CD155 (poliovirus receptor) expressed on target cells
1443 (22-24). DNAM-1’s ability to trigger NK cell responses requires co-expression of the
1444 lymphocyte function-associated antigen 1 (LFA-1), which interacts with the integrins ICAM-1
1445 and ICAM-2 on target cells to form the immunological synapse (25-29). The T cell co-
1446 stimulatory B7 molecules CD80 and CD86 have also been shown to stimulate NK cell-mediated
1447 lysis through a mechanism that is currently undefined, but may depend on a CD28 variant
1448 expressed on NK cells (30-33).
1449 While a subset of the classical MHC-I human leukocyte antigens (HLA) -A, -B, and -C
1450 are ligands to the inhibitory killer immunoglobulin-like receptor (KIR) family, the non-classical
1451 HLA-E and -F interact with aNKR (Reviewed in 34). HLA-E can signal through the inhibitory
1452 C-type lectin-like receptor NKG2A and the activating NKG2E and -C, all of which can form
1453 heterodimers with CD94 (35-37).
1454 It was previously shown that autologous HIV-infected CD4+ T cells are capable of
1455 activating NK cells ex vivo and that these activated NK cells will inhibit further viral replication
1456 (38-40). The HIV accessory proteins Nef and Vpu have well documented effects on expression
1457 of ligands to iNKR on the surface of infected cells. Specifically, both viral proteins contribute to
1458 the internalization and downregulation of HLA-A and -B, abrogating inhibitory signaling
1459 through their cognate iNKR (41-43). As NK cells are directed to kill cells with low expression of
- 64 -
1460 MHC-1 antigens, the downregulation of HLA-A and -B by HIV should make infected cells more
1461 susceptible to NK cell lysis (44). However, a third MHC-1 antigen, HLA-C, and the non-
1462 classical HLA-E antigen remain expressed on the surface of infected cells, where they can
1463 engage the inhibitory KIR2DL1, -2, and -3 and NKG2A/CD94, respectively, to inhibit NK cell
1464 activation (45).
1465 The influence of HIV on iNKR ligand expression and its impact on NK cell activation
1466 and subsequent antiviral responses has been well studied. The contribution of ligands to aNKR,
1467 however, is less well characterized. In this work, we determined whether HIV-I infection can
1468 modulate the expression profile of ligands to aNKRs on infected cells and to identify aNKR
1469 ligands that are preferentially modulated by infection.
- 65 -
1470 3.4: Methods
1471 3.4.1: Ethics Statement
1472 This study was conducted in accordance with the principles expressed in the Declaration
1473 of Helsinki. It was approved by the Institutional Review Boards of the Comité d’Éthique de la
1474 Recheche du Centre Hospitalier de l’Université de Montréal and the Research Ethics Committee
1475 of the McGill University Health Centre - Montreal General Hospital. Informed consent was
1476 obtained for all study participants.
1477 3.4.2: Study population
1478 This study included a total of 17 HIV seronegative donors. The HLA-A, -B, and -C
1479 genotypes of the study participants is presented in Table 1. 11 additional HIV seronegative
1480 donors were included in in the supplemental analysis of viral inhibition.
1481 3.4.3: CD4+ T cell isolation
1482 Peripheral blood mononuclear cells (PBMC) were isolated from blood by density
1483 gradient centrifugation (Ficoll-Paque; Pharmacia, Uppsala, Sweden) and cryopreserved in 10%
1484 dimethyl sulfoxide (DMSO; Sigma-Aldrich, St. Louis, MO) with 90% fetal bovine serum (FBS;
1485 Wisent, Inc. St. Bruno, QC, Canada). CD4+ T cells from 17 HIV seronegative individuals were
1486 positively selected from thawed PBMCs using a commercially available kit, as per
1487 manufacturer’s instructions (Stemcell Technologies, Inc. Vancouver, BC, Canada). Briefly, an
1488 anti-CD4 selection cocktail was added to PBMCs resuspended at 100x106 cells/mL in
1489 Dulbecco’s phosphate buffered saline (dPBS); 2% FBS (Wisent, Inc. St. Bruno, QC, Canada);
- 66 -
1490 1mM EDTA (Sigma-Aldrich, Oakville, ON, Canada) for 15 mins at room temperature (RT). Ab-
1491 bound cells were then mixed with magnetic beads for 10 mins at RT. Cell suspensions were
1492 placed in a magnet for 5 min, after which the supernatant was removed; this process was
1493 repeated for a total of 3 cycles. The purity of the isolated cells was determined by flow cytometry
1494 and averaged 93.6±3.5%.
1495 3.4.4: HIV infection
1496 Following isolation, CD4+ T cells were incubated at 37°C at a concentration of 2x106
1497 cells/mL in RPMI 1640 medium; 10%FBS; 2mM L-glutamine; 100IU/mL penicillin; 100mg/mL
1498 streptomycin (R10; all from Wisent) supplemented with 100IU/mL of recombinant human IL-2
1499 (R10-rhIL-2; Chiron Corp., Emeryville, CA) and activated with 1μg/mL of phytohemagglutinin
1500 (PHA; MP Biomedicals, Santa Ana, CA) for 24 hrs. After activation, cells were washed and
1501 incubated at 37°C in R10-IL2 for 72 hrs. Following this incubation, at least 2x106 cells were
1502 infected with the CCR5-tropic HIV-1JR-CSF at a multiplicity of infection of 0.01 and subsequently
1503 cultured at 37°C at a concentration of 1x106 cells/mL of R10-IL-2 for 7 days. Uninfected CD4+ T
1504 cells (unCD4) were cultured in parallel with their infected counterparts.
1505 3.4.5: Antibody staining and acquisition
1506 On day 7 post-infection, HIV-infected CD4+ T cells (iCD4) and unCD4 were stained with
1507 UV Live/Dead ® Fixable Blue cell stain kit (Thermofisher, Waltham, MA) and then with anti-
1508 CD3-BV785 (OKT3; BioLegend, San Diego, CA) and anti-CD4-BUV737 (SK3; BD Bioscience,
1509 San Jose, CA) in conjunction with fluorochrome conjugated monoclonal antibodies (Abs) with
1510 clone designations shown in parentheses to the following cell surface markers, which were
- 67 -
1511 organized into 4 panels for the purpose of analysis: (Panel 1) ULBP-1-PerCP (170818), ULBP-
1512 2/5/6-APC (165903), ULBP-3-PE (166510), MIC-A-APC (159227), MIC-B-AlexaFluor 700
1513 (236511; all from R&D Systems, Minneapolis, MN), (Panel 2) CD48-PE (BJ40), CD80-BV421
1514 (2D10), CD86-PE-Dazzle594 (IT2.2; all from BioLegend), CD112-AlexaFluor 700 (610603;
1515 R&D), CD155-PE-Cy7 (SKII.4; BioLegend), (Panel 3) ICAM-1-Pacific Blue (HCD54), and
1516 ICAM-2-PE (CBR-1C2/2; both from BioLegend). A final HLA Panel comprised the conjugated
1517 antibodies HLA-E-PE-Cy7 (3D12; BioLegend) and HLA-A2-APC (BB7.2; eBioscience, San
1518 Diego, CA), as well as DT9, an unconjugated Ab against HLA-C (DT9; EMD Millipore,
1519 Billerica, MA). Briefly, cells were resuspended in a 96-well V bottom plate (Sarstedt,
1520 Nümbrecht, Germany) at a concentration of 1x106 cells per 100μL of Dulbecco’s phosphate
1521 buffered saline (dPBS; Wisent) and stained with the Live/Dead reagent. TruStain FcX reagent
1522 (BioLegend) was used to minimize non-specific Fc receptor interactions and cells were stained
1523 with conjugated antibodies from Panel 1, 2, 3, and HLA for 30 min in the dark at room
1524 temperature (RT). Following staining, cells were washed, fixed, and permeabilized using the
1525 reagents in the Fix & Perm Cell fixation and permeabilization kit (Thermofisher), as per
1526 manufacturer’s instruction. Intracellular staining with anti-p24-FITC (KC57; Beckman-Coulter,
1527 Mississauga, ON, Canada) was used to determine the percentage of HIV-infected (iCD4), with
1528 unCD4 providing background values.
1529 iCD4 and unCD4 stained with DT9 were included in the HLA panel and were prepared
1530 as above. They were incubated with the primary Ab on ice for 40 min. After washing, anti-HLA-
1531 C binding was detected with APC-conjugated polyclonal goat F(ab’)2 anti-mouse IgG secondary
1532 Ab (eBioscience) for 20 min on ice. Following this, cells were washed and stained with Abs to
1533 CD3, CD4, and intracellular p24 as previously described. After staining, all cells were washed
- 68 -
1534 and fixed with 2% paraformaldehyde (PFA; Santa Cruz, Dallas TX). Between 400,000 and
1535 600,000 events were acquired on an LSRFortessa x20 (BD) within 24hrs. Unstained, single
1536 stained control (CompBead; BD), fluorescence minus one, and secondary Ab alone controls were
1537 used for multi-colour compensation and gating purposes. Flow cytometric analysis was
1538 performed using FlowJo software version 10 (TreeStar, Ashland OR). All the Abs used and their
1539 manufacturers are listed in Appendix A.
1540 3.4.6: Statistical analysis
1541 Statistical analysis was performed using GraphPad Prism version 6 (GraphPad, San
1542 Diego, CA). Wilcoxon tests or Friedman tests with Dunn’s post-test comparisons were used to
1543 compare the significance of differences in the frequency or mean fluorescence intensity (MFI) of
1544 a cell surface ligand between 2 or more than 2 matched groups, respectively. Spearman
1545 correlation tests were used to determine the significance of correlations between ligand
1546 expression and HIV infection. P-values less than 0.05 were considered significant.
- 69 -
1547 3.5: Results
1548 3.5.1: CD4+ T cell infection characteristics
1549 To investigate the expression of aNKR ligands on CD4+ T cells and the modulatory
1550 effects of HIV-1 infection on aNKR expression, we used 4 Ab panels specific for aNKR ligands.
1551 As the infection protocol requires culturing cells in rhIL-2-containing media, which can
1552 upregulate aNKR ligands, we cultured unCD4 T cells under the same conditions as an internal
1553 control to observe the effects of HIV infection on aNKR profiles irrespective of the effects of IL-
1554 2. The gating strategy in Figure 1A was used to identify T cells as single, live, CD3+
1555 lymphocytes. As illustrated in Figure 1B and C, the expression of CD4 and intracellular p24 – a
1556 measure of HIV-1 infection – was used to delineate 5 different T cell subsets. The first subset
1557 was HIV-uninfected p24-CD4+ T cells that had never been exposed to virus (unCD4s; Fig. 1B).
1558 Similarly, from the population of cells that were cultured with HIV, we identified a subset of
1559 p24-CD4+ T cells that remained HIV-uninfected despite being exposed to virus (euCD4) (Fig.
1560 1C). Total infected p24+ T cells comprise the iCD4T subset. However, consistent with reports
1561 that the HIV-1 viral protein Nef can downregulate surface expression of CD4, two distinct
1562 subsets could be observed within iCD4T (41, 46). We observed an HIV-infected p24+CD4+ T cell
1563 (iCD4+) subset and an HIV-infected p24+CD4- T cells (iCD4-) subset in which cell-surface CD4
1564 was downregulated (Fig. 1B).
1565 3.5.2: Comparison of the expression profiles of ligands to aNKR on HIV-infected CD4+ T
1566 cells that maintain or downregulate CD4
- 70 -
1567 It has been proposed that HIV iCD4- are more productively infected than iCD4+ (46, 47).
1568 In line with this, we observed that iCD4-, compared to iCD4+ cells, have higher expression levels
1569 of p24 on a per cell basis (p≤0.0001, paired t-test, Fig. 1D). To assess the potential phenotypic
1570 implications of these differences in infection kinetics, we compared the frequencies of iCD4+ and
1571 iCD4- that expressed ligands to the Abs in Panels 1, 2, and 3 (Fig. 2). We found that, apart from
1572 cells expressing ULBP-2/5/6, CD86, and ICAM-1, the frequency of iCD4- expressing all other
1573 aNKR ligands detected by these Abs was significantly lower than that of iCD4+ cells (p≤0.008,
1574 for all comparisons, Wilcoxon test).
1575 3.5.3: Comparison of the frequencies of HIV iCD4 and unCD4 T cells expressing ligands to
1576 aNKRs
1577 We next compared the expression profiles of uninfected cells to iCD4T, iCD4+ and iCD4-
1578 cells; Fig. 3). As we observed no significant differences in the expression of each of the aNKR
1579 ligands studied between unCD4 and euCD4 (data not shown), all comparisons are made against
1580 unCD4. We observed that HIV infection altered the frequency of T cells expressing all the aNKR
1581 ligands studied, except CD86. Specifically, we found that a greater frequency of iCD4+ than
1582 unCD4 expressed ULBP-1, ULBP-2/5/6, ULBP-3, MIC-A, CD80, and ICAM-1 (p≤0.0009, for
1583 all comparisons, Friedman with Dunn’s post-test). While a higher frequency of iCD4T than
1584 unCD4 were positive for these aNKR ligands, few of these comparisons reached statistical
1585 significance. Furthermore, the frequency of iCD4- and unCD4 expressing ULBP-1, ULBP-2/5/6,
1586 ULBP-3, MIC-A, and CD80 did not differ significantly (Figure 3A-C). The exception to this was
1587 ICAM-1, where frequencies of all infected subsets (iCD4T, iCD4+, and iCD4-) expressing this
1588 ligand were higher than those of unCD4 (p ≤ 0.0032, for all comparisons). The frequency of
- 71 -
1589 iCD4+ cells expressing MIC-B, CD48, CD112, CD155, and ICAM-2 was not significantly higher
1590 than that of unCD4 (Fig. 3A-C). However, the frequencies of iCD4- cells expressing these aNKR
1591 ligands were less than those of unCD4, although this trend only reached significance for MIC-B
1592 expression (p≤0.0115). Together these results show that HIV infection lead to the upregulated
1593 expression of several aNKR ligands. The lower frequency of several of these ligands on iCD4-
1594 compared to iCD4+ cells suggests that they are downregulated concomitantly with CD4
1595 downregulation.
1596 3.5.4: Comparison of the aNKR ligand expression intensity on iCD4 and unCD4 T cells
1597 To further characterize the extent of ligand modulation in HIV-1-infected CD4+ T cells,
1598 we next assessed potential changes to the per-cell expression of the Panel 1, 2, and 3 aNKR
1599 ligands studied by measuring the mean fluorescence intensity (MFI) of aNKR ligand expression
1600 (Fig. 4). We observed that the MFI of ULBP-1, ULBP-2/5/6, ULBP-3, MIC-A, CD48, CD80,
1601 and CD155 was significantly elevated in iCD4+, but not on iCD4-, compared to unCD4
1602 (p≤0.0232, for all comparisons, Friedman with Dunn’s post-test). The MFI of these ligands was
1603 also elevated in iCD4T, but was only significantly higher than unCD4 for ULBP-3, MIC-A, and
1604 CD80 expression (p=0.0032, p=0.0061, and p=0.0115, respectively). While infection had no
1605 effect on the frequency of T cells expressing CD86, we observed that the expression of this
1606 ligand on iCD4+ and iCD4T was significantly increased, relative to unCD4 (p=0.002 and
1607 p=0.0061, respectively). In contrast, HIV infection was associated with a sustained increase in
1608 the MFI of ICAM-1 on all infected cells, regardless of CD4 expression (p≤0.0077, for all
1609 comparisons). In sum, we observed that HIV infection is associated with changes to the aNKR
1610 ligand expression profile and that changes in the MFI of ligand expression often mirrored the
- 72 -
1611 changes observed in the frequencies of T cells expressing that ligand. Specifically, we found that
1612 compared to HIV-unCD4 the expression of ULBP-1, ULBP-2/5/6, ULBP-3, MIC-A, CD48,
1613 CD80, CD86, CD155, and ICAM-1 was elevated on iCD4+, whereas expression of MIC-B,
1614 CD112, and ICAM-2 was reduced on iCD4-.
1615 3.5.5: Expression profiles of HLA-E, HLA-A2, and HLA-C of iCD4 and unCD4
1616 In addition to the iNKR/aNKR ligand HLA-E, we analysed the expression of two iNKR
1617 ligands, HLA-A2 and HLA-C (Fig. 5). Although HIV Nef can downregulate the expression of
1618 HLA-A and B antigens on the surface of infected cells, cell-surface expression of HLA-E and
1619 HLA-C is maintained (45). While a lower frequency of iCD4-, compared to iCD4+, expressed
1620 HLA-E (p = 0.078, Wilcoxon, Fig. 5A), the frequency and MFI of HLA-E expression on iCD4T
1621 was comparable to that of unCD4 (Fig. 5B and C), consistent with the observation that HLA-E is
1622 retained at the surface of bulk HIV-infected cells (45, 48). Similarly, we found that HIV
1623 infection did not appear to impact HLA-C expression on iCD4T or iCD4-, compared to unCD4
1624 (Fig. 5B and C). Consistent with previous reports, the frequencies of HLA-A2+ iCD4- and iCD4T
1625 were significantly lower than that of unCD4 (p = 0.0061 and p = 0.0372, respectively, Friedman
1626 with Dunn’s post-test) and, while just above the threshold for significance (p=0.0637), the MFI
1627 of HLA-A2 expression on iCD4- was two-fold less than that of unCD4 (49, 50). Overall, our
1628 findings support previous observations that the HIV viral protein Nef downmodulates expression
1629 of HLA-A, while leaving surface expression of HLA-E and HLA-C unmodulated.
- 73 -
1630 3.6: Discussion
1631 Since the surface expression of HLA-A and -B antigens are downregulated over the
1632 course of HIV-infection, it is plausible that modulation of the expression of ligands to aNKRs on
1633 the surface of infected cells may have important regulatory effects on the NK cell responses they
1634 elicit. As such, elucidating HIV-induced regulation of the aNKR ligand expression profile on
1635 infected cells would help further characterize the NK cell stimulating potential of these cells. In
1636 this study, we report that HIV infection is associated with significant changes to the expression
1637 profiles of ligands to aNKRs. We observed two general patterns of HIV-induced ligand
1638 modulation. Specifically, we found that, compared to unCD4, the expression of ULBP-1, ULBP-
1639 2/5/6, ULBP-3, MIC-A, and CD80 was increased exclusively on HIV-infected T cells that
1640 maintained cell-surface CD4 expression (iCD4+), whereas infected cells with downregulated
1641 CD4 (iCD4-) demonstrated reduced expression of MIC-B, CD48, CD112, CD155, and ICAM-2.
1642 Furthermore, ICAM-1 was found to be broadly upregulated on all HIV infected cell subsets
1643 examined.
1644 The influence of HIV infection on the expression of some ligands to aNKR has been
1645 previously investigated in different infection models. While ULBP-1, ULBP-2/5/6, and ULBP-3
1646 are rarely expressed on healthy primary CD4+ T cells, it has been well established that infection
1647 with several laboratory strains of HIV can induce their surface expression (51-54). In contrast,
1648 reports on HIV-mediated regulation of the expression of other NKG2D ligands, i.e. MIC-A and
1649 MIC-B, on CD4+ T cells are conflicting (51, 53, 54). HIV infection induces the loss of surface
1650 expression of CD155 on the Jurkat T cell line, however, this could not be reproduced in bulk
+ 1651 p24 primary HIVNL4-3-infected T cells, which found CD155 to be modestly upregulated (55-57).
- 74 -
1652 Infection of the human monocytic cell line U937 with primary isolates of an Indian HIV-1
1653 subtype C induced the downregulation of CD80 and CD86 (58). Finally, HIV infection has also
1654 been suggested to trigger the cell surface loss of CD48 on infected CD4+ T cells (59). In addition
1655 to these analyses of surface expression, an RNA microarray of primary human CD4+ T cells
1656 infected with the HIV-1-based reporter virus NL4-3 BAL-IRES-HSA found that ULBP-1, CD80,
1657 and ICAM-1 expression was upregulated, compared to mock-infected cells, consistent with our
1658 observations of increased surface expression of these ligands (60). Moreover, the RNA
1659 expression profiles of these ligands did not differ between mock infected cells and uninfected
1660 bystander cells, which are the equivalent of the euCD4 studied here (60).
1661 The results presented here add to this body of work by including aNKR ligands not
1662 previously studied, including CD112 and ICAM-2. Additionally, in vitro HIV-infected CD4+ T
1663 cells can be easily divided into two distinct populations (iCD4+ and iCD4-), depending on their
1664 expression of the CD4 co-receptor. In infected CD4 T cells, CD4 surface expression is
1665 downregulated and internalized by the HIV viral protein Nef (41-43, 46). Regardless, most of the
1666 existing literature assessed the phenotype of bulk p24+ infected cells, regardless of CD4
1667 expression. It has, however, been postulated that iCD4+ and iCD4- differ in the burden and
1668 timing of HIV infection and we also observed that the intensity of staining for the viral capsid
1669 protein p24, which serves as a marker of infection, differed significantly between these two
1670 subsets. HIV-infected CD4+ T cells have been shown in vitro to be capable of activating NK
1671 cells, which in turn can block viral replication (38, 39). The ligands studied here and their
1672 engagement of aNKR likely contribute to anti-HIV NK cell responses and differences in the
1673 expression of these ligands on both subsets of infected cells may alter their ability to activate NK
1674 cells. To address this knowledge gap, we devised a gating strategy that allowed us to examine the
- 75 -
1675 expression profiles of aNKR ligands on both subsets of infected T cells separately, including
1676 iCD4+ and iCD4- cells, and compare them to those of euCD4 and unCD4. As hypothesized
1677 aNKR ligand expression did differ on iCD4+ and iCD4-, with iCD4+ expressing higher levels of
1678 the aNKR ligands studied, with the exception of ULBP-2/5/6 and CD86. Co-culture of NK cells
1679 with autologous iCD4 for 7 days revealed that iCD4+ cells were preferentially depleted, leaving
1680 iCD4- virtually untouched, compared to the co-culture of NK cells with unCD4 (Fig. S1).
1681 Together, these findings suggest that HIV-infected T cells expressing CD4 likely constitute
1682 better stimuli for NK cell activation, as they express higher levels of the aNKR ligands studied
1683 here. Their preferential depletion suggests that they are also targeted more effectively by NK cell
1684 than are iCD4- cells
1685 Additionally, we observed that these two infected T cell subsets differed phenotypically
1686 from unCD4, further emphasizing the ability of HIV infection to modulate aNKR ligand
1687 expression. We found that HIV-induced modulation was ligand specific. The expression of the
1688 stress ligands ULBP-1, ULBP-2/5/6, ULBP-3, and MIC-A and CD80 was increased on iCD4+,
1689 but their expression on iCD4- cells decreased to levels comparable to those observed on unCD4.
1690 On the other hand, HIV-infection did not upregulate the expression of MIC-B, CD112, CD155,
1691 and ICAM-2 on total p24+ HIV-infected cells or iCD4+, compared to unCD4, but reduced
1692 expression of these ligands on iCD4- to levels below those observed on unCD4. This may be a
1693 Nef-dependent event. Nef is responsible for the downregulation of CD4 on these iCD4 and has
1694 previously been demonstrated to influence certain aNKR ligands on a variety of HIV-infected
1695 cell types (41, 43, 46). It is well supported that Nef is required for the selective downregulation
1696 of the MHC-I molecules HLA-A and HLA-B and can also block the trafficking of newly
1697 synthesized MHC-I proteins to the cell surface (45, 49, 61-63). Nef is also instrumental in
- 76 -
1698 inducing the loss of cell surface CD155 in T cell lines and CD80 and CD86 in the human
1699 monocytic cell line U937 (55, 58). While HIV infection has been associated with increased
1700 expression of ULBP-1, ULBP-2, and ULBP-3, Nef expression is associated with the
1701 downregulation of NKG2D ligands and expression of ULBP-1, ULBP-2, and MIC-A increases
1702 more dramatically in cells infected with Nef-deficient virus (51, 54). These findings are
1703 consistent with our observation that the increased expression of ULBP-1, ULBP-2/5/6, ULBP-3,
1704 and MIC-A in iCD4+ is lost in parallel with the Nef-mediated downregulation of CD4.
1705 Furthermore, both Nef expression and blocking of interactions between NKG2D and its ligands
1706 have been shown to reduce NK cell-mediated lysis of HIV-infected Jurkat cells (54).
1707 Considering that downmodulation of activating ligands may thus protect HIV-infected cells from
1708 NK cell targeting, our observation that expression of MIC-B, CD112, CD155, and ICAM-2 is
1709 reduced on iCD4- to levels below those observed on unCD4 is of particular interest. Much like
1710 NKG2D signaling, engagement of the aNKR DNAM-1 by its cognate ligand CD155 has recently
1711 been demonstrated to partially regulate NK-cell mediated lysis of HIV-infected CD4+ T cells, a
1712 process that could be disrupted by the downregulation of CD155 (57). It is therefore plausible
1713 that the downregulation of these ligands to aNKRon the surface of iCD4- may confer a survival
1714 advantage to these cells by dampening their ability to activate NK-cell mediated anti-HIV
1715 responses. Nevertheless, the regulation of these ligands by Nef and other viral proteins is a
1716 complex process and, at this time, the specific contribution of these aNKR ligands to NK
1717 activation and antiviral NK cell responses and the ways in which infection might influence them
1718 remains unclear.
1719 Notably, we found that only ICAM-1 expression was consistently increased across all
1720 infected cell subsets, regardless of CD4 expression. ICAM-1 and ICAM-2 are adhesion
- 77 -
1721 molecules that both contribute to the formation of the immunological synapse between an NK
1722 and target cell, which facilitates NK cell activation (27-29). However, expression of ICAM-1,
1723 but not ICAM-2, was also shown to be required for efficient dendritic cell-mediated HIV
1724 transmission and increases infectivity by almost 10-fold (64, 65). It is possible that the consistent
1725 upregulation of ICAM-1 on all infected cell subsets contributes to improved viral transmission
1726 and may therefore be favorable to viral propagation, despite its effects on NK cell activation.
1727 Furthermore, ICAM-1 was the only aNKR ligand studied whose frequency and intensity of
1728 expression positively correlated with the intensity of p24 staining in both iCD4 subsets and in
1729 CD4T cells (Fig. S2). Nef has been demonstrated to increase p24 levels in HIV-infected primary
1730 CD4+ T cells and ICAM-1 was also observed to be overexpressed in Nef+ HIV-infected
1731 endothelial cells (66, 67). These findings and our observations that HIV induces significantly
1732 different aNKR ligand expression phenotypes on iCD4+ and iCD4- cells, suggest that the activity
1733 of the viral protein Nef may be the underlying factor regulating aNKR ligand expression in HIV-
1734 infected CD4+ T cells. It is also possible that iCD4+ and iCD4- may be at different stages of
1735 infection, as the time from infection to the production of Nef has been shown to vary within
1736 infected CD4+ T cells (68). However, the exact relationship between the timing of Nef
1737 production and CD4 downregulation remains incompletely understood and additional work
1738 characterizing the activity of this viral protein in both iCD4+ and iCD4- and its association with
1739 aNKR ligand expression will be required to confirm these possibilities.
1740 Although our study focuses on these ligands in the context of NK cell activation, it is
1741 important to consider the contribution of several of the ligands studied here to inhibitory
1742 signaling. For example, both CD112 and CD155 are also able to engage the iNKR, T cell
1743 immunoreceptor with immunoglobulin and ITIM motifs (TIGIT) on NK cells (69, 70). The
- 78 -
1744 affinity of TIGIT, which is widely expressed on human NK cells, for CD112 and CD155 is
1745 greater than that of the activating NK cell receptor DNAM-1 (70-72). Furthermore, HLA-E, the
1746 expression of which was not significantly modulated by HIV infection, can signal through both
1747 inhibitory (NKG2A) and activating (NKG2E and -C) members of the NKG2x/CD94 family of
1748 NK cell receptors (35). Finally, HLA-C, which we and others have observed remains expressed
+ 1749 on HIV-infected CD4 T cells, engages the inhibitory killer immunoglobulin receptors
1750 KIR2DL1, -2, and -3, limiting NK cell activation by infected cells (45).
1751 Activation of NK cells both in vitro and in vivo is a complex process that depends on the
1752 balance and potency of the different activating and inhibitory signals transmitted upon
1753 interaction with a potential target cell. While our findings provide the foundation for an
1754 improved understanding of the way HIV infection can impact NK cell activation through the
1755 modulation of the expression of activating ligands, the complete picture of how these changes
1756 contribute to activating signaling and how this might integrate with potential inhibitory signaling
1757 remains to be discovered.
- 79 -
1758 3.7: Figure legends
1759 Figure 1: Gating strategy. Gating strategy identifying live singlet CD3+ T lymphocytes (A).
1760 HIV-uninfected CD4+ T cells were identified from this gate (B), The different subsets of HIV-
1761 infected CD4+ T cells were also identified from this gate (C). (D) Comparison of the mean
1762 fluorescence intensity of p24 expression on HIV-infected p24+CD4+ (iCD4+) and HIV-infected
1763 p24+CD4- (iCD4-) T cells (n=17). A Wilcoxon test was used to determine significance of within
1764 subject differences for the indicated T cell subsets. Each data point represents a separate
1765 infection of CD4+ T cells isolated from one individual. Bar height and error bars represent the
1766 mean and standard deviation for the data set. Significant values are shown; “****” = p< 0.0001.
1767 Figure 2: Frequencies of iCD4+ vs iCD4- expressing aNKR ligands. Comparison of the
1768 frequency of HIV-infected p24+CD4+ (iCD4+) and p24+CD4- (iCD4-) T cells expressing ligands
1769 specific for Panel 1, 2, and 3 Abs. Panel 1 (A) includes ULBP-1 (n=11), ULBP-2/5/6 (n=11),
1770 ULBP-3 (n=11), MIC-A (n=9), and MIC-B (n=9). Panel 2 (B) includes CD48, CD80, CD86,
1771 CD112, and CD155 (n=9, for all). Panel 3 (C) includes ICAM-1 and ICAM-2 (n=9, both). The
1772 percent of cells expressing the indicated ligand is represented on the y-axis. A Wilcoxon test was
1773 used to determine significance of within subject differences for the indicated T cell subsets. Each
1774 data point represents T cells isolated from a separate individual. Significant values are shown;
1775 “**” = p< 0.01; “***” = p< 0.001.
1776 Figure 3: Frequencies of all T cell subsets expressing aNKR ligands. Comparison of the
1777 frequency of HIV-infected p24+CD4+ T cells (iCD4+), p24+CD4- T cells (iCD4-), p24+ T cells
1778 (iCD4T), and HIV-uninfected p24-CD4+ T cells (unCD4) expressing Panel 1, 2, and 3 ligands.
1779 Panel 1 (A) includes ULBP-1 (n=11), ULBP-2/5/6 (n=11), ULBP-3 (n=11), MIC-A (n=9), and
- 80 -
1780 MIC-B (n=9). Panel 2 (B) includes CD48, CD80, CD86, CD112, and CD155 (n=9, for all).
1781 Panel 3 (C) includes ICAM-1 and ICAM-2 (n=9, for both). The percent of cells expressing the
1782 indicated ligand is represented on the y-axis. Friedman (PFriedman) and Dunn’s post (*) tests were
1783 used to determine significance of within subject differences between data sets. Each data point
1784 represents T cells isolated from a separate individual. Bar height and error bars represent the
1785 mean and standard deviation for the data set. Significant values are shown; “*” = p< 0.05; “**” =
1786 p< 0.01; “***” = p< 0.001; “****” = p< 0.0001.
1787 Figure 4: Mean fluorescence intensity of aNKR ligand expressing T cell subsets.
1788 Comparison of the per-cell expression of Panel 1, 2, and 3 ligands to aNKR on HIV-infected
1789 p24+CD4+ T cells (iCD4+), p24+CD4- T cells (iCD4-), total p24+ T cells (iCD4T), and HIV-
1790 uninfected p24-CD4+ T cells (unCD4). Panel 1 (A) includes ULBP-1 (n=11), ULBP-2/5/6
1791 (n=11), ULBP-3 (n=11), MIC-A (n=9), and MIC-B (n=9). Panel 2 (B) includes CD48, CD80,
1792 CD86, CD112, and CD155 (n=9, for all). Panel 3 (C) includes ICAM-1 and ICAM-2 (n=9, for
1793 both). The mean fluorescence intensity (MFI) of the expression of the indicated ligand is
1794 represented on the y-axis. Friedman (PFriedman) and Dunn’s post (*) tests were used to determine
1795 significance of within subject differences between data sets. Each data point represents T cells
1796 isolated from a separate individual. Bar height and error bars represent the mean and standard
1797 deviation for the data set. Significant values are shown; “*” = p< 0.05; “**” = p< 0.01; “***” =
1798 p< 0.001; “****” = p< 0.0001.
1799 Figure 5: Expression profile of the HLA molecules HLA-E, HLA-A2, and HLA-C on CD4+
1800 T cell subsets. Panel A shows the frequency of HIV-infected p24+CD4+ (iCD4+) and p24+CD4-
1801 (iCD4-) T cells expressing HLA-E (n=9), HLA-A2 (n=9), and HLA-C (n=4). The frequency (B)
1802 and mean fluorescent intensity (C) of iCD4+, iCD4-, total HIV-infected p24+ T cells (iCD4T) and
- 81 -
1803 uninfected CD4 (unCD4) cells expressing the indicated ligands for the same study subjects as in
1804 (A). The percent and mean fluorescence intensity (MFI) of cells expressing the indicated ligand
1805 is represented on the y-axis. A Wilcoxon test was used to determine significance of within
+ - 1806 subject differences between iCD4 and iCD4 T cell subsets shown in (A). Friedman (PFriedman)
1807 and Dunn’s post (*) tests were used to determine significance of within subject differences
1808 between the data sets shown in panels (B) and (C). Each data point represents T cells isolated
1809 from a separate individual. Bar height and error bars represent the mean and standard deviation
1810 for the data set. Significant values are shown; “*” = p< 0.05; “**” = p< 0.01; “***” = p< 0.001;
1811 “****” = p< 0.0001.
1812 Supplemental Figure 1: NK cells preferentially target HIV-infected CD4 T cells that
1813 conserve cell-surface CD4 expression. The frequency of HIV-infected (p24+) cells within both
1814 iCD4+ and iCD4- T cell subsets from iCD4 cultured alone (white bars) or co-cultured with NK
1815 cells (grey bars) for 10 days (n=11). Friedman (PFriedman) and Dunn’s post (*) tests were used to
1816 determine significance of within subject differences between data sets. Each data point
1817 represents T cells isolated from a separate individual. Significant values are shown; “*” = p<
1818 0.05; “**” = p< 0.01.
1819 Supplemental Figure 2: The frequency and per-cell expression of ICAM-1 is correlated
1820 with HIV-infection on all infected T cell subsets. Correlation of the frequency (%) (top panel)
1821 and mean fluorescence intensity (MFI) (bottom panel) of ICAM-1 expression with the MFI of
1822 p24 expression in (A) iCD4T, (B) iCD4+, and (C) iCD4- HIV infected T cell subsets. Each data
1823 point represents T cells isolated from a separate individual (n=11). Dotted lines represent 95%
1824 confidence intervals. Significant values are shown.
- 82 -
1825 Table 1. Study population HLA genotypes.
HLA-A HLA-B HLA-C
1 01:01, 03:01 44:03, 49 07:01, 16:01
2 24:01, 26:01 15:01, 57:01 05:01, 06:02
3 03:01, 32:01 13:02, 53:01 04:01, 6
4 01:01, 31:01 49:01 07:01
5 02:01, 03:01 07:02, 27:05 01:02, 07:02
6 02:01, 24:02 38:01, 78:01 07:02
7 02:01 07:02, 57:01 05:01, 06:02
8 03:01, 11:01 40, 57:01 02:02, 04:01
9 01:01, 23:01 14:02, 38:01 08:02, 12:02
10 02:01, 29:02 07:02, 44:03 07:02,16:01
11 02:01, 03:01 27:05, 47:01 02:02, 06:02
12 02:01, 03:01 07:02, 50:01 07:02, 16:02
13 01:01, 02:01 08:01, 40:01 03:02, 07:01
14 02:01 07:02, 08:01 07:01, 07:02
15 02:01, 30:02 07:02, 35:01 04:01, 07:02
16 03:01, 11:01 07:02, 35:01 04:01, 07:02
17 02:01, 24:02 44:02, 01:01 05:01, 08:02
- 83 -
1826 Table 2: Description of the aNKR ligands studied.
Number of Patients Panel Ligand Analyzed ULBP-1 11 ULBP-2/5/6 11 Panel 1 ULBP-3 11 MIC-A 9 MIC-B 9 CD48 9 CD80 9 Panel 2 CD86 9 CD112 9 CD155 9
Panel 3 ICAM-1 11 ICAM-2 9 HLA-E 9 HLA HLA-A2 9 HLA-C 4
a
- 84 -
1827 Figure 1: Gating strategy.
- 85 -
1828 Figure 2: Frequencies of iCD4+ vs iCD4- expressing aNKR ligands.
- 86 -
1829 Figure 3: Frequencies of all T cell subsets expressing aNKR ligands.
- 87 -
1830 Figure 4: Mean fluorescence intensity of aNKR ligand expressing T cell subsets.
a
- 88 -
1831 Figure 5: Expression profile of the HLA molecules HLA-E, HLA-A2, and HLA-C on CD4+
1832 T cell subsets.
a
- 89 -
1833 Figure S1: NK cells preferentially target HIV-infected CD4 T cells that conserve cell-
1834 surface CD4 expression.
- 90 -
1835 Figure S2: The frequency and per-cell expression of ICAM-1 is correlated with HIV-
1836 infection on all infected T cell subsets.
- 91 -
3.8: References
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21. Brown MH, Boles K, van der Merwe PA, Kumar V, Mathew PA, Barclay AN. 1998. 2B4, the natural killer and T cell immunoglobulin superfamily surface protein, is a ligand for CD48. J Exp Med 188:2083-2090. 22. Bottino C, Castriconi R, Pende D, Rivera P, Nanni M, Carnemolla B, Cantoni C, Grassi J, Marcenaro S, Reymond N, Vitale M, Moretta L, Lopez M, Moretta A. 2003. Identification of PVR (CD155) and Nectin-2 (CD112) as cell surface ligands for the human DNAM-1 (CD226) activating molecule. J Exp Med 198:557-567. 23. Pende D, Bottino C, Castriconi R, Cantoni C, Marcenaro S, Rivera P, Spaggiari GM, Dondero A, Carnemolla B, Reymond N, Mingari MC, Lopez M, Moretta L, Moretta A. 2005. PVR (CD155) and Nectin-2 (CD112) as ligands of the human DNAM-1 (CD226) activating receptor: involvement in tumor cell lysis. Mol Immunol 42:463-469. 24. Tahara-Hanaoka S, Shibuya K, Onoda Y, Zhang H, Yamazaki S, Miyamoto A, Honda S, Lanier LL, Shibuya A. 2004. Functional characterization of DNAM-1 (CD226) interaction with its ligands PVR (CD155) and nectin-2 (PRR-2/CD112). Int Immunol 16:533-538. 25. Shibuya A, Campbell D, Hannum C, Yssel H, Franz-Bacon K, McClanahan T, Kitamura T, Nicholl J, Sutherland GR, Lanier LL, Phillips JH. 1996. DNAM-1, a novel adhesion molecule involved in the cytolytic function of T lymphocytes. Immunity 4:573-581. 26. Shibuya K, Lanier LL, Phillips JH, Ochs HD, Shimizu K, Nakayama E, Nakauchi H, Shibuya A. 1999. Physical and functional association of LFA-1 with DNAM-1 adhesion molecule. Immunity 11:615-623. 27. Gross CC, Brzostowski JA, Liu D, Long EO. 2010. Tethering of intercellular adhesion molecule on target cells is required for LFA-1-dependent NK cell adhesion and granule polarization. J Immunol 185:2918-2926. 28. Schleinitz N, March ME, Long EO. 2008. Recruitment of Activation Receptors at Inhibitory NK Cell Immune Synapses. PLOS ONE 3:e3278. 29. Culley FJ, Johnson M, Evans JH, Kumar S, Crilly R, Casasbuenas J, Schnyder T, Mehrabi M, Deonarain MP, Ushakov DS, Braud V, Roth G, Brock R, Kohler K, Davis DM. 2009. Natural killer cell signal integration balances synapse symmetry and migration. PLoS Biol 7:e1000159. 30. Luque I, Reyburn H, Strominger JL. 2000. Expression of the CD80 and CD86 molecules enhances cytotoxicity by human natural killer cells. Hum Immunol 61:721-728. 31. Wilson JL, Charo J, Martin-Fontecha A, Dellabona P, Casorati G, Chambers BJ, Kiessling R, Bejarano MT, Ljunggren HG. 1999. NK cell triggering by the human costimulatory molecules CD80 and CD86. J Immunol 163:4207-4212. 32. Galea-Lauri J, Darling D, Gan SU, Krivochtchapov L, Kuiper M, Gaken J, Souberbielle B, Farzaneh F. 1999. Expression of a variant of CD28 on a subpopulation of human NK cells: implications for B7-mediated stimulation of NK cells. J Immunol 163:62-70. 33. Costa C, Barber DF, Fodor WL. 2002. Human NK cell-mediated cytotoxicity triggered by CD86 and Gal alpha 1,3-Gal is inhibited in genetically modified porcine cells. J Immunol 168:3808-3816. 34. Fauci AS, Mavilio D, Kottilil S. 2005. NK cells in HIV infection: paradigm for protection or targets for ambush. Nat Rev Immunol 5:835-843. 35. Kaiser BK, Barahmand-Pour F, Paulsene W, Medley S, Geraghty DE, Strong RK. 2005. Interactions between NKG2x immunoreceptors and HLA-E ligands display overlapping affinities and thermodynamics. J Immunol 174:2878-2884. 36. Wada H, Matsumoto N, Maenaka K, Suzuki K, Yamamoto K. 2004. The inhibitory NK cell receptor CD94/NKG2A and the activating receptor CD94/NKG2C bind the top of HLA-E through mostly shared but partly distinct sets of HLA-E residues. Eur J Immunol 34:81-90. 37. Lee N, Llano M, Carretero M, Ishitani A, Navarro F, López-Botet M, Geraghty DE. 1998. HLA-E is a major ligand for the natural killer inhibitory receptor CD94/NKG2A. Proceedings of the National Academy of Sciences 95:5199-5204.
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38. Alter G, Martin MP, Teigen N, Carr WH, Suscovich TJ, Schneidewind A, Streeck H, Waring M, Meier A, Brander C, Lifson JD, Allen TM, Carrington M, Altfeld M. 2007. Differential natural killer cell–mediated inhibition of HIV-1 replication based on distinct KIR/HLA subtypes. The Journal of Experimental Medicine 204:3027-3036. 39. Song R, Lisovsky I, Lebouche B, Routy JP, Bruneau J, Bernard NF. 2014. HIV protective KIR3DL1/S1-HLA-B genotypes influence NK cell-mediated inhibition of HIV replication in autologous CD4 targets. PLoS Pathog 10:e1003867. 40. Garcia-Beltran WF, Holzemer A, Martrus G, Chung AW, Pacheco Y, Simoneau CR, Rucevic M, Lamothe-Molina PA, Pertel T, Kim TE, Dugan H, Alter G, Dechanet-Merville J, Jost S, Carrington M, Altfeld M. 2016. Open conformers of HLA-F are high-affinity ligands of the activating NK-cell receptor KIR3DS1. Nat Immunol 17:1067-1074. 41. Chaudhuri R, Lindwasser OW, Smith WJ, Hurley JH, Bonifacino JS. 2007. Downregulation of CD4 by human immunodeficiency virus type 1 Nef is dependent on clathrin and involves direct interaction of Nef with the AP2 clathrin adaptor. J Virol 81:3877-3890. 42. Bresnahan PA, Yonemoto W, Ferrell S, Williams-Herman D, Geleziunas R, Greene WC. 1998. A dileucine motif in HIV-1 Nef acts as an internalization signal for CD4 downregulation and binds the AP-1 clathrin adaptor. Curr Biol 8:1235-1238. 43. Lindwasser OW, Chaudhuri R, Bonifacino JS. 2007. Mechanisms of CD4 downregulation by the Nef and Vpu proteins of primate immunodeficiency viruses. Curr Mol Med 7:171-184. 44. Huard B, Fruh K. 2000. A role for MHC class I down-regulation in NK cell lysis of herpes virus-infected cells. Eur J Immunol 30:509-515. 45. Cohen GB, Gandhi RT, Davis DM, Mandelboim O, Chen BK, Strominger JL, Baltimore D. 1999. The selective downregulation of class I major histocompatibility complex proteins by HIV- 1 protects HIV-infected cells from NK cells. Immunity 10:661-671. 46. Tanaka M, Ueno T, Nakahara T, Sasaki K, Ishimoto A, Sakai H. 2003. Downregulation of CD4 is required for maintenance of viral infectivity of HIV-1. Virology 311:316-325. 47. Lundquist CA, Tobiume M, Zhou J, Unutmaz D, Aiken C. 2002. Nef-mediated downregulation of CD4 enhances human immunodeficiency virus type 1 replication in primary T lymphocytes. J Virol 76:4625-4633. 48. Nattermann J, Nischalke HD, Hofmeister V, Kupfer B, Ahlenstiel G, Feldmann G, Rockstroh J, Weiss EH, Sauerbruch T, Spengler U. 2005. HIV-1 infection leads to increased HLA-E expression resulting in impaired function of natural killer cells. Antivir Ther 10:95-107. 49. Brown A, Gartner S, Kawano T, Benoit N, Cheng-Mayer C. 2005. HLA-A2 down-regulation on primary human macrophages infected with an M-tropic EGFP-tagged HIV-1 reporter virus. J Leukoc Biol 78:675-685. 50. Apps R, Meng Z, Del Prete GQ, Lifson JD, Zhou M, Carrington M. 2015. Relative Expression Levels of the HLA Class-I Proteins in Normal and HIV-Infected Cells. The Journal of Immunology 194:3594-3600. 51. Ward J, Bonaparte M, Sacks J, Guterman J, Fogli M, Mavilio D, Barker E. 2007. HIV modulates the expression of ligands important in triggering natural killer cell cytotoxic responses on infected primary T-cell blasts. Blood 110:1207-1214. 52. Fogli M, Mavilio D, Brunetta E, Varchetta S, Ata K, Roby G, Kovacs C, Follmann D, Pende D, Ward J, Barker E, Marcenaro E, Moretta A, Fauci AS. 2008. Lysis of endogenously infected CD4+ T cell blasts by rIL-2 activated autologous natural killer cells from HIV-infected viremic individuals. PLoS Pathog 4:e1000101. 53. Ward J, Davis Z, DeHart J, Zimmerman E, Bosque A, Brunetta E, Mavilio D, Planelles V, Barker E. 2009. HIV-1 Vpr triggers natural killer cell-mediated lysis of infected cells through activation of the ATR-mediated DNA damage response. PLoS Pathog 5:e1000613. 54. Cerboni C, Neri F, Casartelli N, Zingoni A, Cosman D, Rossi P, Santoni A, Doria M. 2007. Human immunodeficiency virus 1 Nef protein downmodulates the ligands of the activating receptor NKG2D and inhibits natural killer cell-mediated cytotoxicity. J Gen Virol 88:242-250.
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55. Bolduan S, Reif T, Schindler M, Schubert U. 2014. HIV-1 Vpu mediated downregulation of CD155 requires alanine residues 10, 14 and 18 of the transmembrane domain. Virology 464- 465:375-384. 56. Matusali G, Potesta M, Santoni A, Cerboni C, Doria M. 2012. The human immunodeficiency virus type 1 Nef and Vpu proteins downregulate the natural killer cell-activating ligand PVR. J Virol 86:4496-4504. 57. Davis ZB, Sowrirajan B, Cogswell A, Ward JP, Planelles V, Barker E. 2016. CD155 on HIV- Infected Cells Is Not Modulated by HIV-1 Vpu and Nef but Synergizes with NKG2D Ligands to Trigger NK Cell Lysis of Autologous Primary HIV-Infected Cells. AIDS Res Hum Retroviruses doi:10.1089/aid.2015.0375. 58. Chaudhry A, Das SR, Hussain A, Mayor S, George A, Bal V, Jameel S, Rath S. 2005. The Nef protein of HIV-1 induces loss of cell surface costimulatory molecules CD80 and CD86 in APCs. J Immunol 175:4566-4574. 59. Ward J, Bonaparte M, Sacks J, Guterman J, Fogli M, Mavilio D, Barker E. 2007. HIV modulates the expression of ligands important in triggering natural killer cell cytotoxic responses on infected primary T-cell blasts. Blood 110:1207-1214. 60. Imbeault M, Giguère K, Ouellet M, Tremblay MJ. 2012. Exon Level Transcriptomic Profiling of HIV-1-Infected CD4+ T Cells Reveals Virus-Induced Genes and Host Environment Favorable for Viral Replication. PLOS Pathogens 8:e1002861. 61. Schwartz O, Marechal V, Le Gall S, Lemonnier F, Heard JM. 1996. Endocytosis of major histocompatibility complex class I molecules is induced by the HIV-1 Nef protein. Nat Med 2:338-342. 62. Le Gall S, Erdtmann L, Benichou S, Berlioz-Torrent C, Liu L, Benarous R, Heard J-M, Schwartz O. 1998. Nef Interacts with the μ Subunit of Clathrin Adaptor Complexes and Reveals a Cryptic Sorting Signal in MHC I Molecules. Immunity 8:483-495. 63. Swann SA, Williams M, Story CM, Bobbitt KR, Fleis R, Collins KL. 2001. HIV-1 Nef blocks transport of MHC class I molecules to the cell surface via a PI 3-kinase-dependent pathway. Virology 282:267-277. 64. Wang JH, Kwas C, Wu L. 2009. Intercellular adhesion molecule 1 (ICAM-1), but not ICAM-2 and -3, is important for dendritic cell-mediated human immunodeficiency virus type 1 transmission. J Virol 83:4195-4204. 65. Fortin JF, Cantin R, Lamontagne G, Tremblay M. 1997. Host-derived ICAM-1 glycoproteins incorporated on human immunodeficiency virus type 1 are biologically active and enhance viral infectivity. J Virol 71:3588-3596. 66. Malbec M, Sourisseau M, Guivel-Benhassine F, Porrot F, Blanchet F, Schwartz O, Casartelli N. 2013. HIV-1 Nef promotes the localization of Gag to the cell membrane and facilitates viral cell-to-cell transfer. Retrovirology 10:80. 67. Yang F, Liu CQ, Qin XL, Shi JJ. 2010. [HIV-1 Nef regulates ICAM-1 expression on endothelial cells via Erk /Mapk signaling pathway]. Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi 26:44-46. 68. Petravic J, Ellenberg P, Chan ML, Paukovics G, Smyth RP, Mak J, Davenport MP. 2014. Intracellular dynamics of HIV infection. J Virol 88:1113-1124. 69. Stanietsky N, Rovis TL, Glasner A, Seidel E, Tsukerman P, Yamin R, Enk J, Jonjic S, Mandelboim O. 2013. Mouse TIGIT inhibits NK-cell cytotoxicity upon interaction with PVR. Eur J Immunol 43:2138-2150. 70. Stanietsky N, Simic H, Arapovic J, Toporik A, Levy O, Novik A, Levine Z, Beiman M, Dassa L, Achdout H, Stern-Ginossar N, Tsukerman P, Jonjic S, Mandelboim O. 2009. The interaction of TIGIT with PVR and PVRL2 inhibits human NK cell cytotoxicity. Proc Natl Acad Sci U S A 106:17858-17863.
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71. Liu S, Zhang H, Li M, Hu D, Li C, Ge B, Jin B, Fan Z. 2013. Recruitment of Grb2 and SHIP1 by the ITT-like motif of TIGIT suppresses granule polarization and cytotoxicity of NK cells. Cell Death Differ 20:456-464. 72. Wang F, Hou H, Wu S, Tang Q, Liu W, Huang M, Yin B, Huang J, Mao L, Lu Y, Sun Z. 2015. TIGIT expression levels on human NK cells correlate with functional heterogeneity among healthy individuals. European Journal of Immunology 45:2886-2897.
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1837 Chapter 4: Discussion
1838
1839
1840
1841
1842
1843
1844
1845
1846
1847
1848
1849
1850
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1851 4.1: Contributions of this work to the field
1852 The results presented in this thesis are, to our knowledge, the first time that the
1853 expression profile of ligands to activating NK cell receptors (aNKR) on the two HLA null cell
1854 lines, K562 and 721, has been compared head to head. Studies that assessed the expression of
1855 individual aNKR ligands on either cell line are present in the literature. However, the incomplete
1856 characterization of the activating ligand phenotypes of K562 and 721 limited our ability to
1857 explain the differences we previously observed in their ability to activate NK cells. Here we
1858 confirmed that K562 and 721 differ in their expression of aNKR ligands and identified profiles
1859 that were characteristic of each cell line. We also pinpointed specific ligand-receptor pairs that
1860 should be studied further, so as to better understand their individual contribution to NK cell
1861 activation and how they shape the overall stimulatory potential of each cell line.
1862 A significant portion of this thesis is also dedicated to studying the modulation of aNKR
1863 ligand expression by HIV infection. Historically, there is a lack of consensus on how to analyze
1864 HIV-infected CD4 T cells (iCD4) by flow cytometry, with the majority of the literature treating
1865 iCD4 as one homogenous population. However, iCD4 clearly comprise two distinct subsets of
1866 infected cells that can be differentiated by their expression (iCD4+) or not (iCD4-) of cell-surface
1867 CD4. Considering this, the results presented in this thesis offer a unique perspective, as they
1868 directly compare the expression of aNKR ligands on both subsets of infected T cells. While it
1869 has been postulated that both iCD4 subsets differ in stage and productivity of HIV infection, to
1870 our knowledge, our findings provide the first concrete evidence that iCD4+ and iCD4- differ
1871 phenotypically.
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1872 Advances in NK cell biology in recent years have provided the community with new
1873 avenues to address different areas of HIV treatment and cure. The NK cell-mediated antibody-
1874 dependent cellular cytotoxicity has been identified as an important contributor to the protection
1875 offered by effective HIV vaccines (7). Other NK cell responses have also been associated with a
1876 reduction in the risk of infection and with slowed disease progression (101, 103, 325-327).
1877 Recent work has shown promising evidence that NK cell responses can be directed (328, 329).
1878 To adequately direct NK cell responses and to translate this into efficient therapy, however,
1879 requires a holistic characterization of NK receptor signaling and activation. Despite concerted
1880 efforts, the determinants of a specific NK cell response to challenge with allogeneic,
1881 transformed, or virally-infected cells remain unclear. The research presented in this thesis offers
1882 insight into the ligand receptor interactions that may be important in governing individual NK
1883 cell responses and activation as a whole. We believe that this research will assist future studies of
1884 NK cell activation and contribute to eventually harnessing the therapeutic potential of NK cells
1885 in context of HIV infection and beyond.
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1886 4.2: Modulation of cell-surface ligands by HIV Nef
1887 Nef is a myristoylated ~27 kilodalton HIV accessory protein whose production facilitates
1888 the transcription and nuclear export of unspliced or singly-spliced mRNAs encoding Gag, Gag-
1889 Pol, Env, Vpu, Vif, and Vpr (35). Nef is considered to be an HIV early protein and Nef mRNA
1890 can first be detected 20 hours following infection, although a broad distribution of times from
1891 infection to Nef protein production can be observed within HIV-infected CD4+ T cells (330,
1892 331). The Nef protein comprises three general domains: an N-terminal anchor and flexible loop,
1893 a central core, and a C-terminal flexible loop (Reviewed in 332). Both N-terminal myristoylation
1894 and other sequences within the N terminal anchor are crucial for membrane association and, in
1895 conjunction with the central core, are also required for the protein interactions the underlie Nef’s
1896 broad array of functions (333-335). In addition to contributing to viral genomic transcription,
1897 Nef can also influence T cell activation, although these effects are pleiomorphic and may depend
1898 on the cellular context of Nef expression, and increase p24 levels in HIV-infected primary CD4+
1899 T cells, stimulating HIV infectivity (336-338). However, arguably the most well-known function
1900 of Nef is its ability to downregulate the cell-surface expression of the T cell coreceptor CD4
1901 (339). Nef-mediated CD4 downregulation is achieved through the direct interaction of Nef with
1902 the AP-2 clathrin adaptor complex, which induces CD4 endocytosis and degradation in
1903 lysosomes, and requires sequences in the Nef central core that bind to AP-2 and the highly
1904 conserved LL163, 164 and DD174, 175 motifs in the membrane-proximal region of CD4’s
1905 cytoplasmic tail (339-345). This process is facilitated by Vpu, another accessory protein, which
1906 promotes the entry of newly synthesized CD4 into the endoplasmic-reticulum-associated
1907 degradation pathway (11, 58, 59, 346). HIV Nef is also instrumental in the downregulation of the
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1908 additional coreceptors CCR5 and CXCR4, which are required for HIV viral entry (347, 348).
1909 Reduced expression of these entry receptors on the surface of infected T cells promotes viral
1910 replication and persistence by preventing superinfection and subsequent cytotoxicity resulting
1911 from the accumulation of multiple integrated viral genomes, by reducing signaling that could
1912 influence viral transcription or induce apoptosis, and by protecting infected cells from antibody-
1913 mediated cell lysis (45, 346-351).
1914 It is well supported that Nef is required for the selective downregulation of the MHC-I
1915 molecules HLA-A and HLA-B, an ability that relies on sequences in the Nef N-terminal alpha
1916 helix and an interdomain binding site (107, 108, 352-356). The mechanism by which Nef
1917 orchestrates MHC-I downregulation is similar to that of Nef-mediated CD4 downregulation,
1918 although the two are functionally dissociated (353, 355). Nef complexes with the cytoplasmic
1919 tail of MHC-I molecules and the μ1 subunit of the AP-1 clathrin adaptor complex to either
1920 disrupt MHC-I trafficking by re-routing HLA-A and -B from the trans-Golgi network to
1921 lysosomes or induce clathrin-dependent endocytosis (107, 357-363). However, the relative
1922 contribution of each of these pathways to MHC-I modulation remains controversial (Reviewed in
1923 364). Moreover, the cytoplasmic domains of HLA-B appear to be more resistant to Nef-mediated
1924 downregulation and so may not be as reliably downregulated (365, 366). Due to the presence of
1925 unique residues at codons 320 and 327 in the cytoplasmic domain of HLA-C molecules, their
1926 surface expression remains unaltered by Nef activity (108). Maintenance of HLA-C at the
1927 surface of infected cells prevents them from being totally void of MHC-I molecules and of, thus,
1928 being eliminated by missing-self NK cell responses (108). While this is true of laboratory strains
1929 of HIV, as is used in the work presented here, HLA-C can be downregulated by primary HIV
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1930 clones and, paradoxically, this impairs T cell inhibition of viral replication (10). This is
1931 accomplished by the viral accessory protein Vpu, which selectively downregulates HLA-C
1932 through a mechanism that may resemble HLA-A and -B downregulation by Nef (10). It remains
1933 unclear why laboratory strains and primary isolates differ in this manner, although it has been
1934 suggested that viral targeting of HLA-C reflects an adaptation to immune pressure and that
1935 laboratory-adapted virus propagates in vitro in the absence of such pressure (10). Similarly, a
1936 single amino acid substitution in the cytoplasmic tail of the non-classical MHC HLA-E confers
1937 resistance to Nef-mediated downregulation (108). As was initially believed of HLA-C, sustained
1938 HLA-E expression is thought to ensure continued signaling through its iNKR NKG2A,
1939 preventing NK cell-mediated cytolysis of infected cells (108, 187, 367).
1940 It’s clear that a large part of Nef-mediated modulation of surface protein expression
1941 serves to impair anti-HIV responses, including those of NK cells. In line with this, Nef has also
1942 been shown to modulate the expression of certain aNKR ligands and NK cell activation, by
1943 extension. While primary CD4+ T cells respond to HIV infection by increasing surface
1944 expression of the DNAM-1 ligand, CD155, this is attenuated by Nef, which promotes CD155
1945 downregulation and reduces the NK cell-mediated lysis of infected cells (283). In the Jurkat T
1946 cell line, the downregulation of CD155 by Nef shares many similarities with the Nef-mediated
1947 downregulation of MHC-I molecules, including reduced intracellular accumulation of CD155,
1948 requiring similar conserved residues, and being functionally separate from the Nef-mediated
1949 downregulation of CD4 (283). Notably, the downregulation of CD155 appears to be facilitated
1950 by Vpu, which functions with Nef to localize CD155 to perinuclear compartments, and this
1951 ability requires conserved alanine residues in the Vpu transmembrane domain (283, 368). The
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1952 extent of CD155 downregulation, however, may depend on the virus used and on the cell types
1953 infected, as these findings were not observed when using several other HIV strains or when
1954 infecting primary CD4+ T cells (368, 369). Similarly, the expression of NKG2D ligands is
1955 increased by HIV infection, but the surface expression of ULBP-1, ULBP-2, and MIC-A is
1956 downregulated by Nef (144, 176). Downregulation of ULBP-2 and MIC-A appears to be a
1957 conserved function of the laboratory-adapted HIVNL4-3 Nef and from variants obtained from
1958 clinical isolates, however this was not observed for ULBP-1 (176). Additionally, Nef-induced
1959 downregulation of NKG2D ligands likely proceeds through a unique mechanism, as mutations in
1960 residues critical for the downregulation of CD4 and MHC-I molecules had little effect on
1961 NKG2D ligand expression (176). Finally, HIV Nef was observed to upregulate ICAM-1
1962 expression on an endothelial cell line, which requires the protein kinase RNA-like endoplasmic
1963 reticulum kinase (p-ERK) and likely proceeds through extracellular signal-regulated kinase
1964 (ERK)/mitogen-activated protein kinase (MAPK) signaling (370).
1965 Considering these precedents, it seems plausible that, as discussed in Chapter 3, the
1966 different aNKR ligand expression profiles observed on iCD4+ and iCD4- may reflect differences
1967 in the production of Nef. As Nef is responsible for CD4 downregulation and iCD4- are
1968 characterized by their lack of CD4 cell-surface expression, this subset is often considered to be at
+ 1969 a later stage of infection than iCD4 , which have presumably not begun to produce HIV viral
1970 proteins. Considering that Nef expression has not been studied in these individual HIV-infected
1971 T cell subsets, it is possible that Nef protein is being produced in iCD4+, however the sustained
1972 expression of CD4 suggests that Nef is not active in these cells. As such, we speculate that Nef is
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1973 capable of modulating the expression of aNKR ligands and that increased expression of Nef in
1974 iCD4- could explain why aNKR ligand expression on these cells differs from iCD4+.
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1975 4.3: Limitations and future directions
1976 One limitation of this study is that the ligands studied are only considered in the context
1977 of their capacity to bind aNKRs. The potential for several of the ligands studied here to
1978 potentiate inhibitory signaling, as discussed in Chapters 2 and 3, complicates our ability to
1979 extrapolate how ligand expression might influence NK cell activation. Indeed, the obvious
1980 direction for future work in this vein is to address how the aNKR ligand phenotype of the two
1981 HLA-null cell lines studied, K562 and 721, and of iCD4 contributes to NK cell activation. This
1982 can be addressed by additional functional analyses using an NK cell stimulation assay,
1983 previously employed by our lab, to determine the minimal requirements for NK cell activation
1984 (5). Briefly, the percentages of all combinations of cytokine secreting (measured by IFN-γ
1985 expression), chemokine secreting (measured by CCL4 expression), and degranulating (measured
1986 by CD107α expression) NK cells would be assessed by flow cytometry following stimulation
1987 with K562, 721, and iCD4 in the presence or absence of blocking reagents that prevent the
1988 interaction of defined aNKR/ligand pairs. In this context, a significant reduction of a functional
1989 subset in the presence of defined combinations of blocking reagents would be interpreted as
1990 evidence that the targeted aNKR-ligand pairs are required for that NK cell activation stage and
1991 function. Such studies would be particularly useful in confirming that the aNKR ligand profiles
1992 that we observed to be characteristic of K562 or 721 (Figure 1 and 2, Chapter 2) are directly
1993 related to the ability of each HLA-null cell line to activate different numbers and functional
1994 subsets of NK cells.
1995 Furthermore, an experimental framework exists that would allow us to determine which
1996 aNKR-ligand pairs regulate individual stages of NK cell activation, including adhesion, granule
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1997 polarization, actin mobilization, and degranulation. Such experiments would complement the
1998 work described above by contributing more direct data on the aNKR-ligand requirements for
1999 NK-cell mediated cytolysis. Briefly, this method, which was pioneered by Eric O. Long’s group,
2000 involves using Drosophila Schneider cell (SC) 2 cells transfected with individual or
2001 combinations of ligands as target cells (294). This method overcomes the complexity of NK cell
2002 signaling by reducing the multiplicity of receptor-ligand interactions that is observed with
2003 mammalian target cells to one receptor-ligand pair. This approach also dissects total NK cell
2004 responsiveness by using cell-surface expression of CD107α as a marker of degranulation, but
2005 also measures ADCC – for which we have existing in house protocols – and uses confocal
2006 microscopy to analyze the polarization of perforin-containing granules (294). Additionally, the
2007 ability of receptor-ligand pairs to promote adhesion can be observed by measuring their ability to
2008 induce LFA-1 conformational change through what are known as “inside-out” signals (371).
2009 Such methodology has already provided some insight into the importance of certain aNKR-
2010 ligand pairs. Target cells individually expressing ICAM-1 and ULBP-1 were found to induce
2011 granule polarization, but not degranulation, while CD48-2B4 signaling induced weak
2012 polarization and no degranulation (294, 372). Furthermore, concurrent ULBP-1-NKG2D, CD48-
2013 2B4, and ICAM-1-LFA-1 signaling defined a minimal requirement for NK cell cytotoxicity,
2014 although none of these receptor-ligand pairs could induce degranulation individually (371, 373).
2015 However, we believe that valuable data could be gained by using this method to study the ways
2016 in which the aNKR-ligand pairs that were studied here, but absent from previous studies,
2017 influence the distinct stages of NK cell activation, which remains undefined.
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2018 One of the chief findings of this research is that HIV-infected T cells that maintain cell
2019 surface CD4 (iCD4+) or not (iCD4-) differ in their expression of the aNKR ligands studied
2020 (Figure 2 and 4, Chapter 3). Specifically, we observed that iCD4- expressed lower levels of
2021 aNKR ligands, compared to iCD4+. While we can speculate that these differences reflect
2022 variations in the activity of the HIV viral accessory protein Nef, which also downregulates cell
2023 surface CD4, we are limited by the lack of data characterizing these two unique HIV-infected T
2024 cell subsets. Transcriptomic analyses using microarrays and Western blots have previously been
2025 utilized to assess viral gene expression in HIV infected cells (374, 375). A similar approach to
+ - 2026 analyzing iCD4 and iCD4 , could help confirm the mechanism by which HIV modulates aNKR
2027 ligand expression on both infected T cell subsets. Additionally, we observed that iCD4+ were
2028 preferentially targeted by NK cell-mediated inhibition of viral replication (Supplemental Figure
2029 1, Chapter 3). We hypothesize that the susceptibility of iCD4+ to NK cell anti-HIV responses
2030 results from their increased expression of aNKR ligands and, therefore, superior ability to
2031 activate NK cells. However, future experiments that measure NK cell activation induced by
+ - 2032 sorted iCD4 and iCD4 – as could be accomplished by the experimental set up described above
2033 – would help support our theory.
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2034 4.4: Conclusion
2035 The results presented here elaborate a more complete phenotypic characterization of the
2036 cell types commonly used to stimulate NK cell activity, including the HLA-null cell lines K562
2037 and 721 and HIV-infected CD4+ T cells. We confirmed that K562 and 721 each have
2038 characteristic expression profiles of ligands to aNKRs. Furthermore, each profile was capable of
2039 engaging different numbers and subsets of aNKRs, further supporting our hypothesis that
2040 underlying differences in aNKR ligand expression explained the differences in the NK cell
2041 stimulating capacities of K562 and 721 that we previously observed. We have also demonstrated
2042 that HIV infection significantly influenced the expression profile of aNKR ligands of infected
2043 CD4+ T cells in a ligand specific manner. Our head to head comparisons of infected T cells that
2044 maintained CD4 expression (iCD4+) or not (iCD4-) also showed that aNKR ligand expression
2045 differed between these two infected subsets. This finding suggested that differences in the
2046 kinetics of HIV infection may also be important in regulating ligand expression on the surface of
2047 infected cells and would be strengthened by further characterizations of iCD4+ and iCD4-. While
2048 our work offers important insight into how these three cell types might stimulate NK cells,
2049 additional work needs to be done to confirm the contribution of each ligand-aNKR pair to NK
2050 cell activation and anti-HIV responses. Clarifying the mechanisms of NK cell activation,
2051 particularly in the context of HIV infection, will improve our understanding of the mechanisms
2052 of protection against HIV and may identify novel therapeutic targets.
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Appendix A: Antibodies used in the analysis of aNKR ligand expression
Antibody Conjugation Clone Manufacturer Live/Dead Blue/Indo-Violet N/A Thermofisher Anti-human CD3 BV785 OKT3 BioLegend Anti-human CD4 BUV737 SK3 BD Biosciences Anti-human p24 FITC KC57 Beckman-Coulter Anti-human ULBP-1 PerCP 170818 R&D Systems Anti-human ULBP-2/5/6 APC 165903 R&D Systems Anti-human ULBP-3 PE 166510 R&D Systems Anti-human HLA-E PE-Cy7 3D12 BioLegend Anti-human ICAM-1 Pacific Blue HCD54 BioLegend Anti-human HLA-A2 APC BB7.2 eBioscience Anti-human CD80 BV421 2D10 BioLegend Anti-human CD86 PE-Dazzle594 IT2.2 BioLegend Anti-human CD112 Alexa Fluor 700 610603 Cedarlane Anti-human CD155 PE-Cy7 SKII.4 BioLegend Anti-human ICAM-2 PE CBR-1C2/2 BioLegend Anti-human MICA APC 159227 R&D Systems Anti-human MICB Alexa Fluor 700 236511 R&D Systems Anti-human CD48 PE BJ40 BioLegend Anti-human HLA-C N/A DT9 EMD Millipore Anti-human HLA-F N/A 3D11 N/A Recombinant human KIR3DS1-Fc N/A N/A R&D Systems Recombinant human NKp30-Fc N/A N/A R&D Systems Recombinant human NKp44-Fc N/A N/A R&D Systems Recombinant human NKp46-Fc N/A N/A R&D Systems Anti-human IgG (Fc gamma-specific) PE Polyclonal eBioscience F(ab’)2 anti-mouse IgG APC Polyclonal eBioscience
- 109 -
Appendix B: Panel of the ligands studied and their respective activating NK cell receptors
- 110 -
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