Regulation of innate lymphocyte function

National Institute Of Allergy And Infectious Diseases

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Abstract

The CD28-B7 family of receptor-ligand pairs regulates lymphocyte responses through co-stimulation and co-inhibition. It includes checkpoint inhibitors, such as PD-1, which limit anti-tumor and anti-virus T cell responses. CD28 homolog (CD28H, TMIGD2) and B7H7 (HHLA2) have been identified recently as a new receptorligand pair in this family. We have shown that CD28H is a strong activator of primary human NK cells through selective synergy with receptors NKp46 (NCR1) and 2B4 (CD244). Expression of B7H7 on target cells enhanced both natural cytotoxicity and ADCC of NK cells. We engineered a third generation CD28H chimeric antigen receptor (CD28H-CAR) consisting of full-length CD28H linked with the cytoplasmic domains of receptor 2B4 and of the T cell receptor zeta chain (TCRzeta). Remarkably, expression of this CD28H-2B4-TCRzeta CAR in NK cells triggered lysis of B7H7+ HLA-E+ tumor cells by overriding the otherwise dominant inhibition by the HLA-E receptor NKG2A. Furthermore, the substitution of the CD28H extracellular domain with a single-chain CD19 antibody linked to the CD28H-2B4-TCRzeta chimeric cytoplasmic domain, and expression of this NK-tailored CAR in NK cells resulted in lysis of CD19+ B-cell tumor lines. These CAR-NK cells were resistant to the inhibition mediated by HLA-C or by HLA-E on CD19+ B cells. Thus, CD28H is a powerful co-activation receptor of NK cells that broadens their anti-tumor activity and holds promise as a component of NK-based CARs for cancer immunotherapy. 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 erythroleukemia-derived K562 and 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. Both of these cell lines do not express HLA class I and are NK-sensitive. 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). The approach has been validated by the identification of B7H6 (NCR3LG1), the ligand of NK activation receptor NKp30 (NCR3), as the major contributor to lysis of K562 cells. More interesting were other mutations in genes for two master regulators of, respectively, protein glycosylation and half-life of transcription factors. To determine which glycosyl-transferases are responsible for the resistant phenotype of the first mutant, a secondary CRISPR screen in the mutant, using a library of guide RNAs targeting genes that regulate protein glycosylation, will reveal mutations that restore susceptibility to NK cells. Similarly, a secondary CRISPR screen in the other mutant should identify the specific transcription factor, among those targeted for degradation by the regulator, whose half-life regulates sensitivity to NK cells. Such mutants provide valuable information on specific changes that render tumor cells more sensitive or resistant to NK cells.