eprintid: 34759
rev_number: 28
eprint_status: archive
userid: 8115
dir: disk0/00/03/47/59
datestamp: 2024-05-28 13:18:58
lastmod: 2024-06-03 14:33:28
status_changed: 2024-05-28 13:18:58
type: doctoralThesis
metadata_visibility: show
creators_name: Hofman, Tomáš
title: Defining innate immune checkpoints controlling Natural Killer cell recognition of melanoma cells
subjects: ddc-570
divisions: i-140001
adv_faculty: af-14
cterms_swd: Immunologie
abstract: Melanoma, though rare, stands as one of the most lethal forms of skin cancer. Immune checkpoint inhibitors targeting inhibitory receptors on T cells represent an effective therapy that has greatly improved clinical responses against melanoma in a subset of cancer patients. Since NK cells represent the first line of defence against tumors, I used a genome-wide (GW) CRISPR/Cas9 knockout (KO) screen to identify checkpoint candidates on melanoma cells that inhibit human NK cell anti-tumor reactivity. After co-culture with primary NK cells, I observed that melanoma cells deficient in antigen-presenting machinery genes were strongly depleted from the surviving melanoma pool. I determined a crucial role of IFNγ acting as a NK shut off mechanism via NK subset-specific classical or non-classical MHC-I-dependent pathways, resulting in a strong induction of melanoma resistance. I further narrowed down the effect of IFNγ to inhibitory NKG2A and KIR receptors. Accordingly, I confirmed that HLA-E alone fully inhibited NKG2A+ KIR- NK cells, while only partially NKG2A+ KIR+ NK cells. The classical MHC-I molecules mediated the inhibition of NKG2A- KIR+ NK cell subset. The combination of both HLA-E and classical MHC-I were then responsible for the inhibition of NKG2A+ KIR+ NK cells where NKG2A showed dominant inhibitory role over the KIRs. The use of monalizumab, a NKG2A blocking antibody, lirilumab, a KIR2 blocking antibody, and DX9, a commonly used KIR3DL1 blocking antibody, proved sufficient to replicate the observed effects in co-cultures with melanoma HLA KO cells and completely abolished IFNγ-mediated resistance, not only in melanoma cells but also in other tumor entities, rendering them susceptible to NK cell killing.
I further performed a GW CRISPR/Cas9 KO screen in a B2M KO cell line, that excluded the potent role of MHC-I, and showed the sialic acid biosynthesis pathway as another melanoma cell resistant mechanism to NK cell cytotoxicity. The loss of SLC35A1 or TM9SF3 in melanoma cells abolished the expression of ligands for Siglec inhibitory receptors and specifically restored Siglec-9+ NK cell function. 
Thus, combining both aspects of my thesis, generating NKG2A- KIR- Siglec-9- NK cells or using a combination of monalizumab/lirilumab/DX9 antibody treatment combined with a blockade of Siglec-9 could overcome both IFNγ- and sialic acid-mediated tumor cell resistance to NK cell cytotoxicity.
date: 2024
id_scheme: DOI
id_number: 10.11588/heidok.00034759
ppn_swb: 1890524107
own_urn: urn:nbn:de:bsz:16-heidok-347599
date_accepted: 2024-04-23
advisor: HASH(0x5644fa44e460)
language: eng
bibsort: HOFMANTOMADEFININGIN20240423
full_text_status: public
place_of_pub: Heidelberg
citation:   Hofman, Tomáš  (2024) Defining innate immune checkpoints controlling Natural Killer cell recognition of melanoma cells.  [Dissertation]     
document_url: https://archiv.ub.uni-heidelberg.de/volltextserver/34759/1/Tomas%20Hofman%20-%20Thesis.pdf