Inhibitory checkpoints in human natural killer cells: IUPHAR Review 28.

Immune checkpoint inhibitors have revolutionized cancer therapy leading to exceptional success. However, there is still the need to improve their efficacy in non-responder patients. Natural killer (NK) cells represent the first line of defence against tumours, due to their ability to release immunomodulatory cytokines and kill target cells that have undergone malignant transformation. Harnessing NK cell response will open new possibilities to improve control of tumour growth. In this respect inhibitory checkpoints expressed on these innate lymphocytes represents a promising target for next-generation immunotherapy. In this review, we will summarize recent evidences on the expression of NK cells receptors in cancer, with a focus on the inhibitory checkpoint programmed cell death protein 1 (PD-1). We will also highlight the strength and limitations of the blockade of PD-1 inhibitory pathway and suggest new combination strategies that may help to unleash more efficiently NK cell anti-tumour response.

IL15 induces a potent antitumor activity in NK cells isolated from malignant pleural effusions and overcomes the inhibitory effect of pleural fluid.

Natural Killer (NK) cells are capable of recognizing and killing cancer cells and play an important role in tumor immunosurveillance. However, tumor-infiltrating NK cells are frequently impaired in their functional capability. A remarkable exception is represented by NK cells isolated from malignant pleural effusions (PE) that are not anergic and, upon IL2-induced activation, efficiently kill tumor cells. Although IL2 is used in various clinical trials, severe side effects may occur in treated patients. In this study, we investigated whether also other clinical-grade cytokines could induce strong cytotoxicity in NK cells isolated from pleural fluid of patients with primary or metastatic tumors of different origins. We show that PE-NK cells, cultured for short-time intervals with IL15, maintain the CD56bright phenotype, a high expression of the main activating receptors, produce cytokines and kill tumor cells in vitro similarly to those treated with IL2. Moreover, IL15-activated PE-NK cells could greatly reduce the growth of established tumors in mice. This in vivo antitumor effect correlated with the ability of IL15-activated PE-NK cells to traffic from periphery to the tumor site. Finally, we show that IL15 can counteract the inhibitory effect of the tumor pleural microenvironment. Our study suggests that IL15-activated NK cells isolated from pleural fluid (otherwise discarded after thoracentesis) may represent a suitable source of effector cells to be used in adoptive immunotherapy of cancer.

Group 3 innate lymphoid cells regulate neutrophil migration and function in human decidua.

Innate lymphoid cells (ILCs) have a central role in innate defenses against pathogens, lymphoid organogenesis, and tissue remodeling. They have been detected in human decidua, however, their role in this tissue remains unclear. Successful pregnancy requires an early inflammatory phase favoring implantation and tissue remodeling as well as a subsequent regulatory phase to prevent fetal rejection and supporting neoangiogenesis. Here, we show that, during the first trimester of pregnancy, neutrophils infiltrate decidua basalis and are more abundant in normal pregnancy than in spontaneous miscarriages. Decidual neutrophils localize in proximity of NCR+ILC3, which may influence neutrophil migration and survival given their production of CXCL8 and granulocyte macrophage colony-stimulating factor (GM-CSF). Moreover, NCR+ILC3-derived GM-CSF was found to induce the expression of heparin-binding EGF-like growth factor and IL1ra in neutrophils, two proteins/cytokines involved in tissue remodeling and maintenance of pregnancy. Our data suggest that the simultaneous presence of NCR+ILC3 and neutrophils in decidual tissues and their possible cross talk, may have a role in the early phases of pregnancy.

Identification of diverse innate lymphoid cells in human decidua.

Innate lymphoid cells (ILCs) are developmentally related cells that play an important role in innate defenses and tissue remodeling. So far, only natural killer (NK) cells have been identified and functionally characterized in human decidua where they contribute to induction of immune suppression, neo-angiogenesis, and tissue building/remodeling. The presence of other ILC subsets in human decidua has not been yet characterized. Here we identify in human decidua, during early pregnancy, two subsets of decidual group 3 ILC (ILC3), including lymphoid tissue inducer (LTi)-like cells and natural cytotoxicity receptors (NCRs)(+)ILC3 and interferon-(IFN)γ-producing ILC1, different from NK cells. Decidual LTi-like cells produced interleukin -17 (IL-17) and tumor necrosis factor (TNF), while NCR(+)ILC3 released IL-22 and IL-8. Importantly, NCR(+)ILC3 and LTi-like cells established functional interactions with stromal cells. Decidual LTi-like cells differentiated into NCR(+)ILC3, whereas they marginally contributed to NK cell generation. Our data suggest that decidual ILC3 may play a role in innate defenses and in vessel and tissue building, thus contributing to maintenance of pregnancy.

Natural killer alloeffector responses in haploidentical hemopoietic stem cell transplantation to treat high-risk leukemias.

Natural killer (NK) cells, a major cell type of the innate immunity, express surface receptors that regulate potent effector functions such as cytolytic activity and release of cytokines playing a central role in inflammatory response and immunoregulation. In this contribution, we briefly outline the major steps from the discovery of human leukocyte antigen (HLA)-class I-specific inhibitory receptors in humans to recent successful clinical applications in the cure of high-risk leukemias both in adults and in pediatric patients. A central role is played by 'alloreactive' NK cells originated from donor's CD 34(+) cells in eradicating leukemic cells in the setting of T-cell-depleted haploidentical hemopoietic stem cell transplantation. Because alloreactive NK cells play a central role also in preventing graft rejection and graft-vs-host disease, they may represent an ideal tool to treat patients affected by acute high-risk leukemias.

Internalization via Antennapedia protein transduction domain of an scFv antibody toward c-Myc protein.

We constructed a single-chain variable fragment miniantibody (G11-scFv) directed toward the transactivation domain of c-Myc, which is fused with the internalization domain Int of Antennapedia at its carboxyl terminus (a cargo-carrier construct). In ELISA experiments, an EC(50) for binding saturation was achieved at concentrations of G11-scFv-Int(-) of approximately 10(-8) M. Internalization of a fluoresceinated Fl-G11-scFv-Int(+) construct was observed in intact human cultured cells with confocal microscopy. After 5 h of incubation in medium containing 1 microM Fl-G11-scFv-Int(+) or Fl-G11-scFv-Int(-), fluorescence intensity was determined in individual cells, both for cytoplasmic and nuclear compartments: concentration levels of Fl-G11-scFv-Int(+), relative to the extracellular culture medium concentration, were 4-5 times higher in the cytoplasm, 7-8 times higher in the nucleus, and 10 times higher in the nucleoli. In the same experimental conditions, the Fl-G11-scFv-Int(-) construct was 3-4 times more concentrated outside of the cells than inside. Cell membranes kept their integrity after 5 h of incubation. The antiproliferative activity of our miniantibody was studied on HCT116 cells. Incubation with 4 microM G11-scFv-Int(+) for 4 days induced very significant statistical and biological growth inhibition, whereas Int alone was completely inactive. Miniantibodies capable of penetrating cell membranes dramatically broaden the potential for innovative therapeutic agents and attack of new targets.

Purification and HPLC-MS analysis of a naturally processed HCMV-derived peptide isolated from the HEK-293T/HLA-E+/Ul40+ cell transfectants and presented at the cell surface in the context of HLA-E.

A new method for isolation and characterization of peptides presented in the context of the nonclassical human leukocytes antigen (HLA) class I molecule HLA-E was developed. A combination of different chromatographic steps coupled with electrospray mass spectrometry allowed us to detect the presence of small amounts of a naturally processed human Cytomegalovirus (HCMV)-derived peptide isolated from the HEK-293T/HLA-E+/UL40+ transfected cells of from HELA cell line. The peptide sequence was confirmed by tandem mass spectrometry (MS/MS). This approach provides a versatile and sensitive method for direct identification of MHC class I-binding peptides that might be derive from different pathogen or tumor-associated proteins.

Surface receptors and functional interactions of human natural killer cells: from bench to the clinic.

The past 10years have witnessed dramatic progress in our understanding of how natural killer (NK) cells function and their role in innate immunity. Thanks to an array of inhibitory receptors specific for different HLA class I molecules, human NK cells can sense the decrease or loss of even single alleles at the cell surface. This represents a typical condition of a potential danger, i.e. the presence of tumor or virally infected cells. NK cell triggering and lysis of these cells is mediated by several activating receptors and coreceptors that have recently been identified and cloned. While normal cells are usually resistant to NK-mediated attack, a remarkable exception is represented by dendritic cells (DCs). In their immature form they are susceptible to NK-mediated lysis because of the expression of low levels of surface HLA class I molecules. The process of DC maturation (mDCs) is characterized by the surface expression of high levels of HLA class I molecules. Accordingly, mDCs become resistant to NK cells. A recent major breakthrough highlighted the role played by donor NK cells in allogenic bone marrow transplantation to cure acute myeloid leukemias. 'Alloreactive' NK cells derived from donor hematopoietic precursors not only prevented leukemic relapses, but also prevented graft rejection and graft-versus-host disease.

Natural killer cells: a mystery no more.

Recent years have witnessed remarkable progress in our understanding of the molecular mechanism regulating natural killer (NK) cell function. NK cells can sense whether cells have lost the surface expression of major histocompatibility complex (MHC)-class I molecules. The discovery of MHC-class I-specific inhibitory receptors clarified the basis of this discrimination and elucidated the nature of the 'off' signal. However, the receptors responsible for the 'on' signal in the process of natural cytotoxicity remained mysterious until recently. Here, we describe the identification and characterization of such receptors and discuss the emerging implications of these findings in different diseases.

The analysis of the natural killer-like activity of human cytolytic T lymphocytes revealed HLA-E as a novel target for TCR alpha/beta-mediated recognition.

Cytolytic T lymphocytes (CTL) are known to recognize antigen peptides in association with major histocompatibility complex (MHC) class I molecules expressed on target cells. However, a fraction of human CD8(+) CTL has been shown to lyse certain natural killer (NK)-susceptible target cells via still undefined mechanism(s). These CD8(+) T cells, hereafter referred to as NK-CTL, are frequently composed of cells expressing one single TCR Vbeta expansion (different in different individuals), display a memory phenotype and express HLA class I-specific inhibitory NK receptors. Here we show that cell populations or clones of NK-CTL isolated from three healthy donors homogeneously expressed Vbeta16, Vbeta9 and Vbeta3 TCR, respectively. Various clones isolated under limiting dilution conditions from Vbeta16(+) cells of donor 1 displayed identical TCR Vbeta and Valpha rearrangements, thus suggesting a substantial monoclonality of the NK-CTL subset analyzed. NK-CTL lysed a number of NK-susceptible tumor target cells with the exception of those characterized by beta2-microglobulin (beta2m) deficiency. However, the latter targets became susceptible to lysis upon beta2m transfection. Using monoclonal antibodies specific for the relevant TCR Vbeta or beta2m we provide evidence suggesting that target cell lysis by NK-CTL is mediated by the TCR itself upon recognition of beta2m-associated proteins. The cellular distribution of the potential beta2m-associated proteins in susceptible target cells suggested, as a likely candidate for TCR-mediated recognition, the non-classical HLA-E molecule. The use, as target cells, of the murine TAP2-deficient RMA-S cells, either untransfected or transfected with HLA-E, and loaded with an appropriate HLA-E-binding peptide, provided the direct demonstration that HLA-E represents a ligand recognized by the TCR expressed by NK-CTL. This is the first evidence that human TCR alpha/beta can recognize HLA-E molecules, thus revealing a novel type of TCR-mediated recognition, which may offer new insight in immune responses in both normal and disease conditions.

Human natural killer cell function and receptors.

Recent years have witnessed major progress in our understanding of the molecular mechanisms regulating natural killer cell (NK cell) function. These advances stem primarily from the discovery of a number of receptors specific for major histocompatibility complex (MHC) class I and, more recently, of the activating receptors and coreceptors responsible for natural cytotoxicity. Important studies performed over the past year have allowed us to define the evolution of the MHC-specific inhibitory receptors by comparative analysis in different species. The roles of the 'activating natural cytotoxicity receptors', NKG2D and certain coreceptors in the lysis of different tumors have been defined in detail. The mechanism by which the 2B4 coreceptor renders patients with X-linked lymphoproliferative disease unable to control Epstein-Barr virus has been elucidated. Inhibitory receptors identified in NK cells may also be expressed by normal and leukemic myeloid cells, in which they can block cell proliferation and survival. It has also become clear that viruses such as cytomegalovirus have evolved strategies to interfere with NK-cell function to protect themselves from NK-mediated attack.

p75/AIRM1 and CD33, two sialoadhesin receptors that regulate the proliferation or the survival of normal and leukemic myeloid cells.

Inhibitory receptors originally identified in natural killer (NK) cells have also been detected in other leukocyte types, thus suggesting that they may play a more general role in the control of leukocyte function. Here we report data on p75/adhesion receptor molecule 1 (AIRM1), a surface inhibitory receptor of the sialoadhesin family originally identified in NK cells that is also expressed by normal and leukemic myeloid cells. Given the homology between p75/AIRM1 and CD33, we also reanalyzed CD33, a major myeloid marker of still unknown function. We discuss recent data indicating that engagement of p75/AIRM1 or CD33 sharply inhibits the in vitro proliferation/differentiation of CD34+ myeloid precursors induced by stem cell factor and granulocyte-macrophage colony-stimulating factor. Importantly, a similar in vitro inhibitory effect occurs in monocyte/macrophages as well as in chronic or acute myeloid leukemias. While CD33 appears to act via the induction of apoptosis, p75/AIRM1 blocks cell proliferation but does not appear to induce apoptosis. A synergistic effect in the induction of apoptosis has also been documented between antibodies specific for CD33 and the chemotherapic agent etoposide. Taken together, the use of appropriate ligands against CD33 or p75/AIRM1 may represent a new therapeutic tool for treatment of myeloid leukemias or diseases characterized by overwhelming macrophage activation.

Differential disappearance of inhibitory natural killer cell receptors during HAART and possible impairment of HIV-1-specific CD8 cytotoxic T lymphocytes.

BACKGROUND: Highly active antiretroviral therapy (HAART) is associated with a decrease in viral replication to undetectable levels and with an increase in CD4 T lymphocytes. Residual HIV-1 replication occurs together with incomplete recovery of cytotoxic CD8 T lymphocyte (CTL) numbers and function. We sought to determine whether expression of HLA class I-specific inhibitory natural killer receptors (iNKR) on the CTL of patients who had been treated successfully with HAART for 24 months could be involved, at least in part, in residual CTL functional inhibition. METHODS: Two-colour cytofluorometry was used to analyse the expression of six different iNKR including p58.1, p58.2, p70, p140, CD94/NKG2A and LIR1/ILT2 on the CD3, CD8 lymphocytes of eight patients with successful long-term suppression of viral replication before and after 3, 6 and 24 months of HAART. Healthy subjects were analysed as controls. HIV-1-specific cytotoxic activity was determined after 24 months of HAART in the presence and absence of iNKR-masking. RESULTS: No significant reduction of iNKR expression on CD8 T cells was observed by 6 months. Expression of p70 and p140 was inversely correlated with the increasing CD4 numbers. After 24 months CD8 T-lymphocytes expressing p58.1, p58.2, p70, p140 and CD94/NKG2A returned to levels indistinguishable from those of the healthy controls. A significantly increased proportion of CD8 CTL still expressed LIR1/ILT2, a receptor with broad HLA-class I specificity. Functional analysis of freshly separated cells revealed that the disruption of the interaction between LIR1/ILT2 and HLA-class I could partly restore HIV-1-specific lysis. CONCLUSIONS: A decrease in CD3CD8iNKR cells is observed beyond 6 months of HAART. In some patients functional impairment due to LIR1/ILT2 expression may persist even after 24 months of successful HAART.

Differential expression of cytotoxic molecules and killer cell inhibitory receptors in CD8+ and CD56+ cutaneous lymphomas.

Cutaneous lymphomas of the cytotoxic phenotype, including CD8+ and CD56+ lymphomas, have only recently been recognized. To characterize the phenotypic profile of these lymphomas, we investigated the expression of both cytotoxic molecules and killer cell inhibitory receptors by immunohistochemistry techniques. Frozen sections from four CD8+ and from three CD56+ cutaneous lymphomas were stained for the cytotoxicity markers including T-cell restricted intracellular antigen-1, perforin, granzyme B, and for expression of the inhibitory receptors including p58.1, p58.2, p70, p140, CD94, NKG2, and leukocyte immunoglobulin-like receptor (LIR-1). Apart from LIR-1, the CD8+ lymphomas in our series express p70 and p140 from the inhibitory receptors and only one or two of the cytotoxic proteins. The CD56+ lymphomas, on the other hand, express only LIR-1 of the set of inhibitory receptors and the whole panel of cytotoxic antigens. Various subtypes of cytotoxic cutaneous lymphomas (CD8+ and CD56+) differ in regard to their phenotypic and functional profile, which may explain differences in their biological behavior.

Surface expression and function of p75/AIRM-1 or CD33 in acute myeloid leukemias: engagement of CD33 induces apoptosis of leukemic cells.

p75/AIRM-1 is a recently identified inhibitory receptor expressed by natural killer and myeloid cells displaying high homology with CD33. Crosslinking of p75/AIRM-1 or CD33 has been shown to sharply inhibit the in vitro proliferation of both normal myeloid cells and chronic myeloid leukemias. In this study, we analyzed acute myeloid leukemic cells for the expression of p75/AIRM-1. p75/AIRM-1 marked the M5 (11/12) and M4 (2/2) but not the M1, M2, and M3 subtypes according to the French-American-British classification. Cell samples from 12 acute myeloid leukemias were cultured in the presence of granulocyte/macrophage colony-stimulating factor. Addition to these cultures of anti-CD33 antibody resulted in approximately 70% inhibition of cell proliferation as assessed by [(3)H]thymidine uptake or by the recovery of viable cells. Anti-p75/AIRM-1 antibody exerted a strong inhibitory effect only in two cases characterized by a high in vitro proliferation rate. After crosslinking of CD33 (but not of p75/AIRM-1), leukemic cells bound Annexin V and displayed changes in their light-scattering properties and nucleosomal DNA fragmentation, thus providing evidence for the occurrence of apoptotic cell death. Remarkably, when anti-CD33 antibody was used in combination with concentrations of etoposide insufficient to induce apoptosis when used alone, a synergistic effect could be detected in the induction of leukemic cell death. These studies provide the rationale for new therapeutic approaches in myeloid leukemias by using both chemotherapy and apoptosis-inducing mAbs.

Activating receptors and coreceptors involved in human natural killer cell-mediated cytolysis.

Natural killer cells can discriminate between normal cells and cells that do not express adequate amounts of major histocompatibility complex (MHC) class I molecules. The discovery, both in mouse and in human, of MHC-specific inhibitory receptors clarified the molecular basis of this important NK cell function. However, the triggering receptors responsible for positive NK cell stimulation remained elusive until recently. Some of these receptors have now been identified in humans, thus shedding some light on the molecular mechanisms involved in NK cell activation during the process of natural cytotoxicity. Three novel, NK-specific, triggering surface molecules (NKp46, NKp30, and NKp44) have been identified. They represent the first members of a novel emerging group of receptors collectively termed natural cytotoxicity receptors (NCR). Monoclonal antibodies (mAbs) to NCR block to differing extents the NK-mediated lysis of various tumors. Moreover, lysis of certain tumors can be virtually abrogated by the simultaneous masking of the three NCRs. There is a coordinated surface expression of the three NCRs, their surface density varying in different individuals and also in the NK cells isolated from a given individual. A direct correlation exists between the surface density of NCR and the ability of NK cells to kill various tumors. NKp46 is the only NCR involved in human NK-mediated killing of murine target cells. Accordingly, a homologue of NKp46 has been detected in mouse. Molecular cloning of NCR revealed novel members of the Ig superfamily displaying a low degree of similarity to each other and to known human molecules. NCRs are coupled to different signal transducing adaptor proteins, including CD3 zeta, Fc epsilon RI gamma, and KARAP/DAP12. Another triggering NK receptor is NKG2D. It appears to play either a complementary or a synergistic role with NCRs. Thus, the triggering of NK cells in the process of tumor cell lysis may often depend on the concerted action of NCR and NKG2D. In some instances, however, it may uniquely depend upon the activity of NCR or NKG2D only. Strict NKG2D-dependency can be appreciated using clones that, in spite of their NCR(dull) phenotype, efficiently lyse certain epithelial tumors or leukemic cell lines. Other triggering surface molecules including 2B4 and the novel NKp80 appear to function as coreceptors rather than as true receptors. Indeed, they can induce natural cytotoxicity only when co-engaged with a triggering receptor. While an altered expression or function of NCR or NKG2D is being explored as a possible cause of immunological disorders, 2B4 dysfunction has already been associated with a severe form of immunodeficiency. Indeed, in patients with the X-linked lymphoproliferative disease, the inability to control Epstein-Barr virus infections may be consequent to a major dysfunction of 2B4 that exerts inhibitory instead of activating functions.

Dendritic cells efficiently cross-prime HLA class I-restricted cytolytic T lymphocytes when pulsed with both apoptotic and necrotic cells but not with soluble cell-derived lysates.

Dendritic cells (DC) have been shown to efficiently present antigen to CD8(+) cytolytic T cells (CTL) when pulsed with apoptotic cells as a source of cell-derived antigen. Such cross-priming could not be detected by the use of necrotic cells, while conflicting results have been reported for cell-derived soluble lysates. In this study, we reinvestigated this issue by using autologous Epstein-Barr virus-transformed lymphoblastoid cell lines (LCL) as a source of antigen to pulse monocyte-derived DC. Both autologous and HLA-mismatched allogeneic LCL have been used either in the form of apoptotic or of necrotic cells or of soluble cell lysates. At day 7, DC were co-cultured with the autologous CD8(+) lymphocyte fraction for an additional 9 days, in the presence of exogenous IL-2 (added after 48 h). At the end of the culture period, CD8(+) CTL efficiently lysed autologous LCL only when they had been co-cultured with DC pulsed with necrotic or apoptotic cells. That an efficient cross-priming of autologous CD8(+) cells could be induced by DC pulsed with apoptotic or necrotic LCL but not with cell lysates was further demonstrated in assays of IFN-gamma production in response to short-term re-stimulation of CD8(+) cells with LCL. In addition, LCL-specific CD8(+) cells could specifically lyse autologous DC that had been pulsed with LCL-derived antigens, further suggesting that DC presented exogenous antigens on HLA class I molecules.