Regulation of innate lymphocyte function

National Institute Of Allergy And Infectious Diseases

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Abstract

Dominant inhibitory receptors for HLA class I (HLA-I) endow NK cells with high intrinsic responsiveness, a process termed licensing or education, but hinder their ability to kill HLA-I+ tumor cells. Cancer immunotherapy with adoptive transfer of NK cells must overcome inhibitory signals by such receptors to promote elimination of HLA-I+ tumor cells. As proof of concept, we have shown that a chimeric antigen receptor (CAR) can be engineered to overcome inhibition by receptors for HLA-I and to promote lysis of HLA-I+ tumor cells by CAR-NK cells. The design of this NK-tailored CAR (NK-CAR) relied on the potent NK cell activation induced by the synergistic combination of NK receptors CD28H (CD28 homolog, TMIGD2) and 2B4 (CD244, SLAMF4). An NK-CAR consisting of the single-chain fragment variable (scFv) of a CD19 antibody, the CD28H transmembrane domain, and the fusion of CD28H, 2B4, and TCRzeta signaling domains was compared to a third-generation T-cell CAR with a CD28-41BB-TCRzeta signaling domain. The NK-CAR delivered stronger activation signals to NK cells and induced more robust tumor cell lysis. Furthermore, such CAR-NK cells could overcome inhibition by HLA-E or HLA-C expressed on tumor cells. Therefore, engineering of CAR-NK cells that could override inhibition by HLA-I in patients undergoing cancer immunotherapy is feasible. This approach offers an attractive alternative to more complex strategies, such as genetic editing of inhibitory receptors in CAR-NK cells or treatment of patients with a combination of CAR-NK cells and checkpoint blockade with antibodies to inhibitory receptors. A significant benefit of inhibition-resistant NK-CARs is that NK cell inhibition would be overcome only during contact with targeted tumor cells and that HLA-I on healthy cells would continue to maintain NK cell responsiveness through licensing. The power of genome wide CRISPR/Cas9 screens is the ability to identify genes that contribute, even partially, to either sensitivity or resistance to any selection applied. We performed genome-wide screens with two cell lines that are sensitive to lysis by NK cells and identified genes for proteins that confer either sensitivity to, or resistance against NK cell cytotoxicity. The EBV-transformed 721.221 cells were transfected with Cas9 and subjected to genome-wide CRISPR screens for reduced or augmented lysis by primary NK cells during long-term co-cultures at very low effector to target ratios. As 721.221 cells do not express classical HLA class I they are particularly sensitive to NK cells. Selection for mutant cells that escaped lysis by NK cells resulted in enrichment of guide RNAs targeting genes that confer vulnerability (e.g., ligand of an NK activation receptor). Mutant cells that gained sensitivity to NK cells and were killed more rapidly could be identified by the depletion of guide RNAs targeting genes that confer resistance (e.g., cancer-promoting genes). Mutations in a gene that regulates protein glycosylation resulted in resistance (or reduced sensitivity to NK cells. This gene encodes SPPL3, a protease that cleaves the transmembrane region of several glycosyl transferases, thereby reducing their accumulation in the Golgi apparatus. In the absence of functional SPPL3 these glycosyl transferases are more abundant in the Golgi apparatus. To determine what kind of glycans were responsible for the resistant phenotype, the SPPL3 mutant was subjected to a secondary CRISPR screen using a library of guide RNAs targeting genes that regulate protein glycosylation. If an increase in overall glycosylation is responsible for the resistance, one would expect to find multiple genes for glycosyl transferases that partially restore sensitivity. In contrast, the results identified one type of complex N-glycans that was the main contributor to the resistance of 721.221-SPPL3-KO cells. It is generally thought that IL-12 produced by dendritic cells in response to pathogens is the initial signal for IFN-gamma production by NK cells. Using single-cell measurements, cell sorting, and mouse lines deficient in IL-12, IL-23, type I interferon receptor, and the IL-18 receptor, we showed that a subset of bone marrow-derived DCs characterized by low expression of MHC class II stimulate IFN-gamma production by NK cells. Expression of Toll-like Receptor 4 on DCs but not NK cells was required for IFN-gamma production. Supernatant of LPS-activated DCs was sufficient to induce IFN-gamma production by NK cells, independently of IL-12, IL-23, and IL-18. We conclude that a previously unsuspected pathway for DC-mediated IFN-gamma production by NK cells exists, which is independent of the well-known cytokines that stimulate NK cells.