Harnessing NK cells for cancer immunotherapy: immune checkpoint receptors and chimeric antigen receptors

Natural killer (NK) cells, key antitumor effectors of the innate immune system, are endowed with the unique ability to spontaneously eliminate cells undergoing a neoplastic transformation. Given their broad reactivity against diverse types of cancer and close association with cancer prognosis, NK cells have gained considerable attention as a promising therapeutic target for cancer immunotherapy. NK cell-based therapies have demonstrated favorable clinical efficacies in several hematological malignancies but limited success in solid tumors, thus highlighting the need to develop new therapeutic strategies to restore and optimize antitumor activity while preventing tumor immune escape. The current therapeutic modalities yielding encouraging results in clinical trials include the blockade of immune checkpoint receptors to overcome the immune-evasion mechanism used by tumors and the incorporation of tumor-directed chimeric antigen receptors to enhance NK cell antitumor specificity and activity. These observations, together with recent advances in the understanding of NK cell activation within the tumor microenvironment, will facilitate the optimal design of NK cell-based therapy against a broad range of cancers and, more desirably, refractory cancers.     

Recruitment of activation receptors at inhibitory NK cell immune synapses

Natural killer (NK) cell activation receptors accumulate by an actin-dependent process at cytotoxic immune synapses where they provide synergistic signals that trigger NK cell effector functions. In contrast, NK cell inhibitory receptors, including members of the MHC class I-specific killer cell Ig-like receptor (KIR) family, accumulate at inhibitory immune synapses, block actin dynamics, and prevent actin-dependent phosphorylation of activation receptors. Therefore, one would predict inhibition of actin-dependent accumulation of activation receptors when inhibitory receptors are engaged. By confocal imaging of primary human NK cells in contact with target cells expressing physiological ligands of NK cell receptors, we show here that this prediction is incorrect. Target cells included a human cell line and transfected Drosophila insect cells that expressed ligands of NK cell activation receptors in combination with an MHC class I ligand of inhibitory KIR. The two NK cell activation receptors CD2 and 2B4 accumulated and co-localized with KIR at inhibitory immune synapses. In fact, KIR promoted CD2 and 2B4 clustering, as CD2 and 2B4 accumulated more efficiently at inhibitory synapses. In contrast, accumulation of KIR and of activation receptors at inhibitory synapses correlated with reduced density of the integrin LFA-1. These results imply that inhibitory KIR does not prevent CD2 and 2B4 signaling by blocking their accumulation at NK cell immune synapses, but by blocking their ability to signal within inhibitory synapses.     

Unravelling natural killer cell function: triggering and inhibitory human NK receptors

Natural killer (NK) cells represent a highly specialized lymphoid population characterized by a potent cytolytic activity against tumor or virally infected cells. Their function is finely regulated by a series of inhibitory or activating receptors. The inhibitory receptors, specific for major histocompatibility complex (MHC) class I molecules, allow NK cells to discriminate between normal cells and cells that have lost the expression of MHC class I (e.g., tumor cells). The major receptors responsible for NK cell triggering are NKp46, NKp30, NKp44 and NKG2D. The NK-mediated lysis of tumor cells involves several such receptors, while killing of dendritic cells involves only NKp30. The target-cell ligands recognized by some receptors have been identified, but those to which major receptors bind are not yet known. Nevertheless, functional data suggest that they are primarily expressed on cells upon activation, proliferation or tumor transformation. Thus, the ability of NK cells to lyse target cells requires both the lack of surface MHC class I molecules and the expression of appropriate ligands that trigger NK receptors.     

The actin cytoskeleton controls the efficiency of killer Ig-like receptor accumulation at inhibitory NK cell immune synapses

Killer cell Ig-like receptors (KIRs) are MHC class I-specific receptors expressed in NK and T lymphocytes. KIR antagonism of activation signals occurs at the immune synapse between the effector and target cells. The processes that regulate clustering of KIR are not well defined. We have expressed KIR-GFP receptor chimeras in two human NK-like lines, YTS and NK92. In this study, we show that the frequency of KIR enrichment at the synapse was decreased for a KIR that lacks a portion of the cytoplasmic tail. Strikingly, blocking actin polymerization with a high dose of cytochalasin D also substantially decreased clustering of KIR as well as KIR-induced clustering of HLA-C-GFP in target cells. However, the effect of inhibiting actin polymerization was only clearly evident at the earlier time points after cell mixing, and eventually clustering of KIR and HLA-C occurred independently of actin remodeling. Although treatment with anti-LFA-1 also decreased conjugate formation, the frequency of KIR clustering remained normal within the population of conjugates that did form, suggesting that the effect of cytochalasin D is not solely through LFA-1. Collectively, these data suggest that the actin cytoskeleton and the cytoplasmic tail of KIR regulate the efficiency by which KIR accumulates at inhibitory NK cell synapses.     

Harnessing autophagy for cell fate control gene therapy

We hypothesized that rapamycin, through induction of autophagy and promotion of an antiapoptotic phenotype, would permit lentiviral (LV)-based transgene delivery to human T-Rapa cells, which are being tested in phase II clinical trials in the setting of allogeneic hematopoietic cell transplantation. Manufactured T-Rapa cells were exposed to supernatant enriched for a LV vector encoding a fusion protein consisting of truncated CD19 (for cell surface marking) and DTYMK/TMPKΔ, which provides “cell-fate control” due to its ability to phosphorylate (activate) AZT prodrug. LV-transduction in rapamycin-treated T-Rapa cells: (1) resulted in mitochondrial autophagy and a resultant antiapoptotic phenotype, which was reversed by the autophagy inhibitor 3-MA; (2) yielded changes in MAP1LC3B and SQSTM1 expression, which were reversed by 3-MA; and (3) increased T-Rapa cell expression of the CD19-DTYMKΔ fusion protein, despite their reduced proliferative status. Importantly, although the transgene-expressing T-Rapa cells expressed an antiapoptotic phenotype, they were highly susceptible to cell death via AZT exposure both in vitro and in vivo (in a human-into-mouse xenogeneic transplantation model). Therefore, rapamycin induction of T cell autophagy can be used for gene therapy applications, including the CD19-DTYMKΔ cell-fate control axis to improve the safety of T cell immuno-gene therapy.     

Kinoids: a novel generation of specific immune therapy against cytokines

The abnormal cytokine release in the stromal microenvironment of pathologic tissues, contributes to the pathogenesis of viral infections such as AIDS, cancer and auto-immune diseases. Neovacs developed therapeutic vaccines, named Kinoids, which induce anti-cytokine Antibodies. Kinoids are non toxic but immunogenic cytokine derivatives. Kinoid immunizations induce high titre of neutralizing Abs to the corresponding cytokine, is well tolerated and experimentally effective. In transgenic mice expressing huTNFalpha, the TNFalpha kinoid decreases clinical signs of Rheumatoid Arthritis and in mice challenged with syngenic CT26 tumor cell line huVEGF kinoid inhibits lung metastases. After validation by clinical trials, kinoid vaccines could represent a second generation of specific immune therapy to be used to combat ectopic cytokines.     

Protein kinase C regulates expression and function of inhibitory killer cell Ig-like receptors in NK cells

The inhibitory killer cell Ig-like receptors (KIR) negatively regulate NK cell cytotoxicity by activating the Src homology 2 domain-containing protein tyrosine phosphatases 1 and 2 following ligation with MHC class I molecules expressed on normal cells. This requires tyrosine phosphorylation of KIR on ITIMs in the cytoplasmic domain. Surprisingly, we have found that KIR3DL1 is strongly and constitutively phosphorylated on serine and weakly on threonine residues. In this study, we have mapped constitutive phosphorylation sites for casein kinases, protein kinase C, and an unidentified kinase on the KIR cytoplasmic domain. Three of these phosphorylation sites are highly conserved in human inhibitory KIR. Functional studies of the wild-type receptor and serine/threonine mutants indicated that phosphorylation of Ser(394) by protein kinase C slightly suppresses KIR3DL1 inhibitory function, and reduces receptor internalization and turnover. Our results provide evidence that serine/threonine phosphorylation is an important regulatory mechanism of KIR function.     

Interleukin-15 enhances natural killer cell cytotoxicity in patients with acute myeloid leukemia by upregulating the activating NK cell receptors

Interleukin-15 (IL-15) has a major role in NK-cell homeostasis. Modulation of the relative frequency and expression intensity of the NK-cell receptors by IL-15 may increase NK cell-mediated cytotoxicity in cancer patients. We investigated the receptor repertoire and measured NK-cell activity in newly diagnosed AML patients and evaluated the ex vivo effects of IL-15. The expression of the activating NK cell receptors was significantly decreased in the AML patients compared to that in NK cells of healthy donors. When NK cells obtained from AML patients were cultured with IL-15, expression of the activating receptors was significantly upregulated compared to pre-culture levels. Concomitantly, cytotoxic activity of NK cells against autologous leukemic blasts increased following IL-15 stimulation. This IL-15 induced increase in activity was blocked by neutralizing antibodies specific for the NK cell activating receptors. These pre-clinical data support the future use of IL-15 for NK cell- based therapies for AML patients.     

Immunotherapy in gliomas: limitations and potential of natural killer (NK) cell therapy

Malignant gliomas (MGs) are deadly brain tumors with a median survival after resection, radiotherapy and chemotherapy of only 12 months. The natural immunosuppressive state of MG patients and the locally restricted growth of MGs render this neoplasm an excellent target for immunotherapy. Consequently, several failed attempts were made to treat MGs with immune cells. Recent preclinical experimental studies, however, demonstrate that natural killer (NK) cells can kill MGs and therefore hold promise in immunotherapy of MGs. This review describes the experimental and clinical evidence that support the potential of NK cells in therapy of MGs as well as the limitations to NK therapy. Finally, we propose strategies that could circumvent mitigating factors and enhance NK cell therapy for MG patients.     

The evolution of natural killer cell receptors

Natural killer (NK) cells are immune cells that play a crucial role against viral infections and tumors. To be tolerant against healthy tissue and simultaneously attack infected cells, the activity of NK cells is tightly regulated by a sophisticated array of germline-encoded activating and inhibiting receptors. The best characterized mechanism of NK cell activation is “missing self” detection, i.e., the recognition of virally infected or transformed cells that reduce their MHC expression to evade cytotoxic T cells. To monitor the expression of MHC-I on target cells, NK cells have monomorphic inhibitory receptors which interact with conserved MHC molecules. However, there are other NK cell receptors (NKRs) encoded by gene families showing a remarkable genetic diversity. Thus, NKR haplotypes contain several genes encoding for receptors with activating and inhibiting signaling, and that vary in gene content and allelic polymorphism. But if missing-self detection can be achieved by a monomorphic NKR system why have these polygenic and polymorphic receptors evolved? Here, we review the expansion of NKR receptor families in different mammal species, and we discuss several hypotheses that possibly underlie the diversification of the NK cell receptor complex, including the evolution of viral decoys, peptide sensitivity, and selective MHC-downregulation.     

Virus-driven evolution of natural killer cell receptors

Natural killer (NK) cells can mount an immediate response against viral infection, secreting cytokines and killing virus-infected cells. However, viruses have devised strategies to avoid immune detection. Here, we discuss NK cell recognition of viruses and propose that viruses may provide the evolutionary pressure causing the diversification of the NK cell receptors.     

Harnessing features of adaptive NK cells to generate iPSC-derived NK cells for enhanced immunotherapy

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Harnessing biocompatible chemistry for developing improved and novel microbial cell factories

White biotechnology relies on the sophisticated chemical machinery inside living cells for producing a broad range of useful compounds in a sustainable and environmentally friendly way. However, despite the impressive repertoire of compounds that can be generated using white biotechnology, this approach cannot currently fully replace traditional chemical production, often relying on petroleum as a raw material. One challenge is the limited number of chemical transformations taking place in living organisms. Biocompatible chemistry, that is non-enzymatic chemical reactions taking place under mild conditions compatible with living organisms, could provide a solution. Biocompatible chemistry is not a novel invention, and has since long been used by living organisms. Examples include Fenton chemistry, used by microorganisms for degrading plant materials, and manganese or ketoacids dependent chemistry used for detoxifying reactive oxygen species. However, harnessing biocompatible chemistry for expanding the chemical repertoire of living cells is a relatively novel approach within white biotechnology, and it could potentially be used for producing valuable compounds which living organisms otherwise are not able to generate. In this mini review, we discuss such applications of biocompatible chemistry, and clarify the potential that lies in using biocompatible chemistry in conjunction with metabolically engineered cell factories for cheap substrate utilization, improved cell physiology, efficient pathway construction and novel chemicals production.     

Characterization of a novel killer cell lectin-like receptor (KLRH1) expressed by alloreactive rat NK cells

NK cells have the ability to recognize and kill MHC-mismatched hemopoietic cells. In the present study, strain-specific differences in the rat NK allorecognition repertoire were exploited to generate Abs against receptors that may be involved in allogeneic responses. A mAb termed STOK9 was selected, and it reacted with subsets of NK cells and NKR-P1(+) T cells from certain rat strains possessing highly alloreactive NK cells. The STOK9(+) NK subset was broadly alloreactive and lysed Con A lymphoblast targets from a range of MHC-mismatched strains. The mAb STOK9 precipitated a 75-kDa dimeric glycoprotein from NK lysates. Expression cloning revealed that each monomer consisted of 231 aa with limited homology to other previously characterized killer cell lectin-like receptors (KLRs). This glycoprotein therefore constitutes a novel KLR branch, and it has been termed KLRH1. A gene in the central region of the natural killer gene complex on rat chromosome 4 encodes KLRH1. A mouse homolog appears to be present as deduced from analyses of genomic trace sequences. The function of KLRH1 is unknown, but it contains an immunoreceptor tyrosine-based inhibitory motif, suggesting an inhibitory function. The MHC haplotype of the host appears to influence KLRH1 expression, suggesting that it may function as an MHC-binding receptor on subsets of NK cells and T lymphocytes.     

Hybrid-cell vaccines for cancer immune therapy

Hybrid cells generated by fusing allogenic-presenting cells, such as dendritic cells, with tumor cells are a new tool in cancer immunotherapy which are designed to enhance the immunogenicity of antigenic tumors by presenting the whole spectrum of tumor-associated antigens, by providing the co-stimulatory molecules required for T-cell activation, and by the expression of allogenic MHC molecules for recruitment and activation of T-cell help. This approach has been successfully tested in animal models as well as in clinical phaseI/II trials with various tumors. Besides clinical repsonses, induction of tumor-specific cytolytic T-cells were observed. The electrofusion protocol described here has the advantage of high fusion efficiency, high hybrid-cell viability, as well as high reproducibility, and can be used for various tumor cell types after minor adjustments are made to the instrument settings in order to process large numbers of dendritic cells with consistent efficiencies.