Phenotypic and Functional Characterization of Natural Killer Cell Receptors in Human and Rhesus macaque
by Md Zahidul Hasan
Date of Examination:2024-01-15
Date of issue:2024-02-05
Advisor:Prof. Dr. Lutz Walter
Referee:Prof. Dr. Lutz Walter
Referee:Prof. Dr. Holger Reichardt
Referee:Prof. Dr. Rüdiger Behr
Referee:Prof. Dr. Dieter Kube
Referee:Prof. Dr. Wolfram-Hubertus Zimmermann
Referee:Prof. Dr. Uwe Groß
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EnglishActivating killer immunoglobulin-like receptors (KIRs) cannot transmit signals directly but through signaling motifs. Since human activating KIRs lack signaling molecules, their transmembrane lysine residue pairs with adaptor proteins like ITAM signaling motif-containing DAP12. KIR2DL4 is an exceptional activating KIR, possesses a transmembrane arginine residue and pairs with ITAM-containing FcRγ (encoded by the FCER1G gene) instead of DAP12. Interestingly, the formation of Rhesus macaque (Rh) activating KIR genes is thought to be due to recombination between the KIR3D and KIR2DL4 genes. Hence, Rh activating KIRs retain a short cytoplasmic tail and transmembrane arginine residue. In this Thesis, Paper I demonstrated that the FcRγ binds to the Rh activating KIR, thereby stabilizing activating KIR expression and transducing signals that lead to cell activation once activating KIR-expressing cells interact with their cognate ligands (Mamu-A1*001 and -A1*011). Human NKG2A/C receptors play a crucial role in the regulation of Natural Killer (NK) cell function by recognizing HLA-E. A significant proportion of human NK cells primarily express inhibitory CD94/NKG2A receptors over stimulatory CD94/NKG2C. Human cytomegalovirus (HCMV) exposure often induces NKG2C expression and downregulates NKG2A, leading to HCMV-driven distinct and long-lived NK cell subsets with adaptive features, known as HCMV-associated adaptive NK cells. In contrast to human single-copy NKG2C gene, three NKG2C genes (NKG2C-1/C-2/C-3) have been identified in Rh, but their phenotypes and functional characteristics on NK cells remained unknown due to the lack of suitable antibodies. Paper II described hybridoma clones (4A8 and 7B1) that precisely react with Rh CD94/NKG2C-1 and CD94/NKG2C-2 but no reaction with Rh CD94/NKG2A or CD94/NKG2C-3 receptors. A significant increase of Rh NKG2C-1/C-2+ (4A8+) and a decrease of NKG2A+ (4A8-Z199+) NK cells were identified in Rhesus cytomegalovirus+ (RhCMV+) animals compared to RhCMV- animals applying antibody clones 4A8 and Z199. RhCMV+ animal NK cells revealed similar characteristics to those of HCMV-associated adaptive NK cells, such as downregulation of NKG2A, IL12RB2, ZBTB16, and SH2D1B but upregulation of IFNG genes and an increase in KIR expression, confirming the clear detection of RhCMV-associated adaptive NK cells. Notably, small-scale RNA-seq analysis on 4A8+ Rh NK cells showed co-transcription of all four Rh NKG2 genes. In the functional assay, Rh stimulatory NKG2C receptor-expressing cells increased degranulation and IFN-γ expression, whereas NKG2A+ cells were inhibited upon interaction with their cognate ligand, Mamu-E. These stimulatory receptors also exhibited stronger binding avidity to Mamu-E-peptide complex than the inhibitory NKG2A. The expansion of stimulatory Rh NKG2C receptors and the adaptive characteristics of NK cells in response to RhCMV infection suggest a potential beneficial side effect of CMV-driven adaptation persisting in Rh similar to that in human. NK cells are often inhibited via the NKG2A/HLA-E interaction, whereas adaptive NK cell subsets could shift this inhibition to activation due to the increase in NKG2C and subsequent interaction of NKG2C/HLA-E. HLA-E plays an important role in modulating NK cell function during infection. In contemporary COVID-19 circumstances, understanding the SARS-CoV-2 infection and the response of NK cells is essential. Paper III depicted that experimental SARS-CoV-2 infection increases HLA-E expression on the cell surface of human primary lung epithelial tissue sections and lung epithelial cell lines. By analyzing the entire SARS-CoV-2 proteome, spike protein (SP) or non-structural protein 13 (NSP13)-derived peptides (YLQPRTFLL or VMPLSAPTL) were predicted to bind to HLA-E. An increase in HLA-E expression was observed upon transfecting the SP or NSP13-endocing plasmid DNA into cells. Addition of the exogenous YLQPRTFLL peptide exhibited a dose-dependent significant increase in HLA-E expression on HLA-E+ TAP-deficient cells, similar to the HLA-G leader peptide sequences. Mutations in the SP targeting YLQPRTFLL resulted in a decrease in HLA-E expression, confirming its impact on HLA-E. In the functional studies, upon recognition of SP-induced HLA-E ligands, NKG2A+ and NKG2C+ NK cells were inhibited and activated, respectively, during co-incubation of SP-transfected cells with PBMCs (isolated from the donors that either possess conventional or adaptive NK cells). There was a suppression of conventional NK cell activity via inhibition of NKG2A+ NK cells, whereas adaptive NK cells exhibited an increase in degranulation and cytokine production due to their high proportion of NKG2C+ NK cells. By blocking SP-induced HLA-E expression, the inhibitory effect of NKG2A was abolished as observed by an increase in early apoptosis on target cells, indicating the ‘‘missing-self’’-mediated NK cell activation. Thus, SP-induced HLA-E regulates NK cell function, while the NKG2C+ adaptive NK cells of an individual may enhance the immune response to SARS-CoV-2 infection, as shown in Paper III. Overall, my study on NK cell receptors and their function collectively advances our understanding of innate immunity and holds potential implications for immunotherapy. Moreover, understanding the NK cell receptors in the Rh model may enhance our capacity to exploit human NK cells for therapeutic purposes and enhance our knowledge of human immune responses. Also, investigation into SARS-CoV-2 infection and NK cell function provides critical information for the ongoing fight against the COVID-19 pandemic, offering a comprehensive view of how adaptive NK cells might contribute to antiviral immunity.
Keywords: Natural Killer cell; Killer immunoglobulin-like receptor; C-type lectin-like receptor; NKG2A; NKG2C; HLA-E; Adaptive NK cell; Adaptor protein; SARS-CoV-2; Cytomegalovirus