NK cells: immune cross-talk and therapeutic implications

Abstract

Increased evidence of cross-talk between NK cells and other immune cells has enhanced the possibilities of exploiting the interplay between the activation and inhibition of NK cells for immunotherapeutic purposes. The battery of receptors possessed by NK cells help them to efficiently detect aberrant and infected cells and embark on the signaling pathways necessary to eliminate them. Endogenous expansion of NK cells and their effector mechanisms are under exploration for enhancing adoptive immunotherapy prospects in combination with immunostimulatory and cell-death-sensitizing treatments against cancer, viral infections and other pathophysiological autoimmune conditions. Various modes of NK cell manipulation are being undertaken to overcome issues such as relapse and graft rejections associated with adoptive immunotherapy. While tracing the remarkable properties of NK cells and the major developments in this field, we highlight the role of immune cooperativity in the betterment of current immunotherapeutic approaches.

Figure 1. NKG2D signaling

NKG2D is a disulfide-bonded hexameric homodimer, which is associated with the adaptors DAP10 at the cell membrane in humans, or with both DAP10 and/or DAP12 in mice. In mice, NKG2D exists as two isoforms: NKG2D-L contains 13 more amino acids in its cytoplasmic tail than the NKG2D-S isoform. An arginine residue within the transmembrane region of NKG2D associates with the aspartate residue within the transmembrane domain of the homodimeric DAP10 signaling adaptor. NKG2D-S can pair with either DAP10 or DAP12, while NKG2D-L can pair only with DAP10 as the extra 13 amino acids in its tail prevent its binding with DAP12. The phosphorylation of the signal motif present in the cytoplasmic domain of DAP10, enables it to bind either the p85 subunit of PI3K or the adaptor Grb2. The binding of Grb2 induces downstream phosphorylation of Vav1 and PLC-γ2. The adaptor protein CrkL, the small GTPase Rap1 and the scaffolding protein IQGAP1 play important roles downstream of PI3K. Such signaling leads to calcium influx, actin reorganization and degranulation. DAP12 triggers immunoreceptor tyrosine-based activation motif-mediated PI3K signaling via Syk and ZAP70 protein tyrosine kinases and can activate cytokine secretion as well as cytotoxicity.

Figure 2. NK signaling cross-talk

Upon engagement of a ligand by the inhibitory receptor, the tyrosine residue of inhibitory motif ITIM is phosphorylated so as to recruit the lipid phosphatase SHIP or the tyrosine phosphatase SHP-1 or SHP-2. These dephosphorylate Vav of the activation pathway and block the subsequent signaling for activation. Alternatively, phosphorylation of the adaptor protein Crk by Abl1 disrupts the activation complex. Cbl may also inhibit phosphorylation of Vav leading to a blockade of the activation pathways. The ITAM-containing residues are phosphorylated by the Src family protein tyrosine kinases to bind Syk and ZAP tyrosine kinases. At this point, intracellular signaling molecules such as PLC-γ and the nucleotide exchange factors of the Vav family are recruited by NK cells depending on their stage of maturation. A cascade of reactions follows which induces degranulation using Ca²⁺ ions, NF-κB for transcription of cytokine and chemokine genes or LAT-mediated actin reorganization and degranulation. The recruitment of Grb to the DAP10 signaling motif triggers the downstream phosphorylation of Vav1, ultimately leading to actin reorganization and degranulation through a series of reactions. The ITSM of the CD244 receptor molecules is recognized by SAP (SH2D1A), which is a cytoplasmic SH2 domain-containing adaptor protein. Tyrosine phosphorylation of the ITSM by SAP leads to the activation of NK cells via a series of reactions. ITAM: Immunoreceptor tyrosine-based activation motif; ITIM: Immunoreceptor tyrosine-based inhibitory motif; ITSM: Immunoreceptor tyrosine-based switch motif; LAT: Linker of activated T cell.

Figure 3. NK–dendritic cell cross-talk

The TLR ligands secreted by an infected cell are captured by the DCs, the professional antigen-presenting cells. This stimulates the DCs to produce type I interferon through the IRF-3 and IRF-7-mediated JAK–STAT pathway. IL-2, IL-15 and other cytokines presented by the DC to naive NK cells, together with type I interferons, activate the naive NK cells and stimulate their activation and proliferation. NK cells, in turn, regulate the functioning of DCs by eliminating immature DCs with low MHC expression through various effector pathways. Mature DCs with an upregulated expression of MHC are not affected by such pathways; instead, they undergo further activation. DC: Dendritic cell; DNAM: DNAX accessory molecule; TLR: Toll-like receptor; TRAIL: TNF-related apoptosis-inducing ligand.

Figure 4. NK–macrophage cross-talk

Following microbial infection macrophages secrete proinflammatory cytokines IL-12, IL-18, TNF-α, type I interferon and CCR7, which activate NK cells. Polarization of M0 and M2 macrophages to M1 leads to enhanced NK activation. Activated NK cells eliminate M0 and M2 macrophages as they have low levels of MHC expression on their surface. M1s escape this attack owing to upregulated MHC on their surface, but in certain situations can be inhibited by NK cell-secreted IL-10. However, in most cases macrophage activation factors secreted by activated NK cells lead to further activation of macrophages.

Figure 5. NK–T cell cross-talk

T cells secrete IL-2 and IL-15, whose roles in NK activation are well established. IFN-γ and IL-12 secreted by NK cells help in T cell activation and their proliferation into CTL and Th. IL-10 and DNAM-1 secreted by NK cells inhibit T-cell response. NK cells can also contribute to the resolution of T-cell response via the deletion of CTL. CTL: Cytolytic T lymphocytes; DNAM: DNAX accessory molecule; Th: Helper T cells.

Figure 6. NK cell-based strategies for cancer therapy

Allogeneic NK cell transfers are considered advantageous for cancer therapy owing to the better graft-versus-tumor effects and lower graft-versus-host disease effects encountered in patients undergoing such transplants. The lymphocytes harvested from the donor are purified to obtain NK cells while removing the T cells that usually bring about the graft-versus-host disease. Purified NK cells, when transplanted into the recipient, provide long-term remission. Hematopoietic stem cells (HSC) are preferred over bone marrow stem cells as they can bring the cells of the immune system to prechemotherapy levels. Such a strategy provides a good clinical outcome, especially with haploidentical HSC transfers. The rate of graft-versus-tumor, however, is better with allogeneic NK cell transfers as compared with HSC transfers. External administration of NK cell-stimulatory cytokines enhances endogenous NK cell activation. While IFN-γ and GM-CSF have been approved for administration in cancer patients, studies are underway for IL-12, IL-18, IL-21 and IL-23, as well as for genetically modified NK cells. The combined effects of IL-2 and IL-21 have shown promising results by improving immune cross-talk, but the individual administration of these cytokines as well as IL-12 has been shown to produce toxic effects. Various drugs that increase tumor cell expression of ligands for NK cell-activating receptors are currently under investigation currently. The different modes by which these act mostly involve upregulation of NKG2D ligands on tumor cells or the induction of immunostimulatory cytokines. Some drugs such as thalidomide have direct antitumor effects, while others such as bortezomib, a proteasome inhibitor, sensitize tumors to apoptosis and upregulate TRAIL receptors, as well as increasing the expression of NKG2D ligands on tumor cells. Clinical trial with a blocking killer cell immunoglobulin like receptors monoclonal antibody, 1-7F9, which recognizes KIR2DL1, 2 and 3 and thus blocks the inhibitory signaling by almost all MHC class I alleles, is under development. Mild hyperthermal stress brings about increased apoptosis of tumor cells and leads to an increase in NKG2D-mediated cytotoxic killing by NK cells. Heat treatment of NK cells close to the physiological range brings about cellular changes that lead to NK cell activation. HSCT: Hematopoietic stem cell transplantation.