Natural Cytotoxicity Receptors NKp46 Research Articles

On the humanization of VHHs: Prospective case studies, experimental and computational characterization of structural determinants for functionality.

The humanization of camelid-derived variable domain heavy chain antibodies (VHHs) poses challenges including immunogenicity, stability, and potential reduction of affinity. Critical to this process are complementarity-determining regions (CDRs), Vernier and Hallmark residues, shaping the three-dimensional fold and influencing VHH structure and function. Additionally, the presence of non-canonical disulfide bonds further contributes to conformational stability and antigen binding. In this study, we systematically humanized two camelid-derived VHHs targeting the natural cytotoxicity receptor NKp30. Key structural positions in Vernier and Hallmark regions were exchanged with residues from the most similar human germline sequences. The resulting variants were characterized for binding affinities, yield, and purity. Structural binding modes were elucidated through crystal structure determination and AlphaFold2 predictions, providing insights into differences in binding affinity. Comparative structural and molecular dynamics characterizations of selected variants were performed to rationalize their functional properties and elucidate the role of specific sequence motifs in antigen binding. Furthermore, systematic analyses of next-generation sequencing (NGS) and Protein Data Bank (PDB) data was conducted, shedding light on the functional significance of Hallmark motifs and non-canonical disulfide bonds in VHHs in general. Overall, this study provides valuable insights into the structural determinants governing the functional properties of VHHs, offering a roadmap for their rational design, humanization, and optimization for therapeutic applications.

Affinity Maturation of the Natural Ligand (B7-H6) for Natural Cytotoxicity Receptor NKp30 by Yeast Surface Display.

In recent years, the development of bispecific antibodies (bsAbs) has experienced tremendous progress for disease treatment, and consequently, a plethora of bsAbs is currently scrutinized in clinical trials. Besides antibody scaffolds, multifunctional molecules referred to as immunoligands have been developed. These molecules typically harbor a natural ligand entity for the engagement of a specific receptor, while binding to the additional antigen is facilitated by an antibody-derived paratope. Immunoligands can be exploited to conditionally activate immune cells, e.g., natural killer (NK) cells, in the presence of tumor cells, ultimately causing target-dependent tumor cell lysis. However, many ligands naturally show only moderate affinities toward their cognate receptor, potentially hampering killing capacities of immunoligands. Herein, we provide protocols for yeast surface display-based affinity maturation of B7-H6, the natural ligand of NK cell-activating receptor NKp30.

Abstract 3436: Novel multifunctional tetravalent CD38 NKp46 FLEX NK࣪ engagers actively target and kill multiple myeloma cells

Abstract Given that CD38 is a clinically validated target for NK cell mediated cytotoxicity in multiple myeloma, we sought to leverage our FLEX NKTrademark platform to create a NK engager antibody targeting CD38. FLEX NKTrademark is a proprietary platform for production of tetravalent multifunctional NK engager antibodies with a novel FLEX linker to allow for simultaneous binding of both the targeted cancer cells and NK cells. NK engagement and activation is mediated through a binder directed against the natural cytotoxicity receptor NKp46. With the FLEX NKTrademark scaffold, we created two novel tetravalent NK cell engagers targeting CD38, one with a wild type (WT) Fc and one with a mutant null Fc. Both CD38 NKp46 engagers showed dose dependent binding to CD38 expressing multiple myeloma cell lines MM1S and KMS11 and no binding to a CD38 knock out MM.1S cell line. Both engagers also bound multiple myeloma cell lines with ~ 2-fold higher mean fluorescence intensity than anti-CD38 monoclonal antibody (mAb)or daratumumab alone. Epitope mapping studies for our anti-CD38 mAb using alanine scanning mutagenesis showed that amino acid S274 on CD38, critical for binding to daratumumab, is not important for our anti-CD38 antibody binding to CD38, suggesting a distinct epitope detected by our antibody. Interestingly, the NKp46 monoclonal antibody alone without a functional Fc induced peripheral blood NK cell cytolysis of the multiple myeloma cells, consistent with a prior report that NKp46 plays a key role in NK-cell mediated killing of myeloma cells. Both CD38 NKp46 engagers showed further enhanced dose dependent NK cell redirected cytolysis and degranulation against multiple myeloma cells compared to anti-NKp46 monoclonal antibody. The CD38 NKp46 engagers demonstrated higher cytotoxicity than the anti-CD38 binder mAb or daratumumab alone. The CD38 NKp46 engager with the WT Fc is more potent in induction of TNF-α and IFN-ɣ production compared to daratumumab and the engager with the mutant Fc. No IL-1β or IL-6 was induced by the engagers or daratumumab. Daratumumab treatment resulted in NK cell fratricide, while no fratricide was observed with the CD38-NKp46 NK engager with the mutant Fc. In peripheral blood mononuclear cell hemato-toxicity studies depletion of monocytes and NK cells were observed with daratumumab but no depletion was observed with the CD38 NKp46 engager with the mutant Fc. These results suggest that the CD38 NKp46 engagers have a favorable NK cell engager profile for targeting CD38 expressing multiple myeloma that’s distinct from daratumumab. Citation Format: Liang Lin, Hao-Ming Chang, Cecilia Nakid, Stanley Frankel, Dennis Wu, Jean Kadouche, Daniel Teper, Ofer Mandelboim, Jean-Christophe Bories, Antonio Arulanandam, Wei Li. Novel multifunctional tetravalent CD38 NKp46 FLEX NK࣪ engagers actively target and kill multiple myeloma cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3436.

The Natural Cytotoxicity Receptor NKp44 (NCR2, CD336) Is Expressed on the Majority of Porcine NK Cells Ex Vivo Without Stimulation.

Natural killer (NK) cells have been studied extensively in humans and mice for their vital role in the vertebrate innate immune system. They are known to rapidly eliminate tumors or virus infected cells in an immune response utilizing their lytic properties. The natural cytotoxicity receptors (NCRs) NKp30 (NCR3), NKp44 (NCR2), and NKp46 (NCR1) are important mediators of NK-cell cytotoxicity. NKp44 expression was reported for NK cells in humans as well as in some non-human primates and found exclusively on activated NK cells. Previously, no information was available on NKp44 protein expression and its role in porcine lymphocytes due to the lack of species-specific monoclonal antibodies (mAbs). For this study, porcine-specific anti-NKp44 mAbs were generated and their reactivity was tested on blood and tissue derived NK cells in pigs of different age classes. Interestingly, NKp44 expression was detected ex vivo already on resting NK cells; moreover, the frequency of NKp44+ NK cells was higher than that of NKp46+ NK cells in most animals analyzed. Upon in vitro stimulation with IL-2 or IL-15, the frequency of NKp44+ NK cells, as well as the intensity of NKp44 expression at the single cell level, were increased. Since little is known about swine NK cells, the generation of a mAb (clone 54-1) against NKp44 will greatly aid in elucidating the mechanisms underlying the differentiation, functionality, and activation of porcine NK cells.

Evaluating the serum levels of natural killer cells and its activating receptors in patients with Endometriosis

Natural killer cells (NK) play an importantrole causing implantation and proliferation of ectopic endometrial tissue inpatients with endometriosis. The study aims to assess the effect of endometriosison the frequency of NK cells, its activation and inhibition in the peripheral bloodof endometriotic patients.Methods:Sixty four patients are divided into twogroups: 31 healthy controls and 33 endometriosis patients divided into twosubgroups: Group (A) mild stage endometriosis and group (B) moderate and severestage endometriosis. NK cells were identified by monoclonal antibodies surfacestaining for (CD56, CD16), natural cytotoxicity receptor Nkp46, inhibitoryreceptor CD159a, perforin and granzyme using flow cytometry.  Results: There was a statistical significant increase inthe total percent of NK CD56+ in endometriosis group compared to healthy group (p=0.027). There was statisticalsignificant decrease in percentage of NKp46 in endometriosis group compared to thehealthy group (p<0.0001).  No significant difference between differentstages of the disease & control cases in CD159a, perforin and granzyme I. There was negativeCorrelation between Total NK percent and Nkp46 activity.Conclusions:there is a significantincrease in NK cells and decrease in NKp46 in endometriosis patients. Whileno difference in CD159, perforin and granzyme between patients and normal women

Grabbing the Bull by Both Horns: Bovine Ultralong CDR-H3 Paratopes Enable Engineering of 'Almost Natural' Common Light Chain Bispecific Antibodies Suitable For Effector Cell Redirection.

A subset of antibodies found in cattle comprises ultralong CDR-H3 regions of up to 70 amino acids. Interestingly, this type of immunoglobulin usually pairs with the single germline VL gene, V30 that is typically very conserved in sequence. In this work, we have engineered ultralong CDR-H3 common light chain bispecific antibodies targeting Epidermal Growth Factor Receptor (EGFR) on tumor cells as well as Natural Cytotoxicity Receptor NKp30 on Natural Killer (NK) cells. Antigen-specific common light chain antibodies were isolated by yeast surface display by means of pairing CDR-H3 diversities following immunization with a single V30 light chain. After selection, EGFR-targeting paratopes as well as NKp30-specific binders were combined into common light chain bispecific antibodies by exploiting the strand-exchange engineered domain (SEED) technology for heavy chain heterodimerization. Biochemical characterization of resulting bispecifics revealed highly specific binding to the respective antigens as well as simultaneous binding to both targets. Most importantly, engineered cattle-derived bispecific common light chain molecules elicited potent NK cell redirection and consequently tumor cell lysis of EGFR-overexpressing cells as well as robust release of proinflammatory cytokine interferon-γ. Taken together, this data is giving clear evidence that bovine bispecific ultralong CDR-H3 common light chain antibodies are versatile for biotechnological applications.

Natural Killer Cells Generated From Human Induced Pluripotent Stem Cells Mature to CD56brightCD16+NKp80+/-In-Vitro and Express KIR2DL2/DL3 and KIR3DL1.

The differentiation of human induced pluripotent stem cells (hiPSCs) into T and natural killer (NK) lymphocytes opens novel possibilities for developmental studies of immune cells and in-vitro generation of cell therapy products. In particular, iPSC-derived NK cells gained interest in adoptive anti-cancer immunotherapies, since they enable generation of homogenous populations of NK cells with and without genetic engineering that can be grown at clinical scale. However, the phenotype of in-vitro generated NK cells is not well characterized. NK cells derive in the bone marrow and mature in secondary lymphoid tissues through distinct stages from CD56brightCD16- to CD56dimCD16+ NK cells that represents the most abandoned population in peripheral blood. In this study, we efficiently generated CD56+CD16+CD3- NK lymphocytes from hiPSC and characterized NK-cell development by surface expression of NK-lineage markers. Hematopoietic priming of hiPSC resulted in 31.9% to 57.4% CD34+CD45+ hematopoietic progenitor cells (HPC) that did not require enrichment for NK lymphocyte propagation. HPC were further differentiated into NK cells on OP9-DL1 feeder cells resulting in high purity of CD56brightCD16- and CD56brightCD16+ NK cells. The output of generated NK cells increased up to 40% when OP9-DL1 feeder cells were inactivated with mitomycine C. CD7 expression could be detected from the first week of differentiation indicating priming towards the lymphoid lineage. CD56brightCD16-/+ NK cells expressed high levels of DNAM-1, CD69, natural killer cell receptors NKG2A and NKG2D, and natural cytotoxicity receptors NKp46, NKp44, NKp30. Expression of NKp80 on 40% of NK cells, and a perforin+ and granzyme B+ phenotype confirmed differentiation up to stage 4b. Killer cell immunoglobulin-like receptor KIR2DL2/DL3 and KIR3DL1 were found on up to 3 and 10% of mature NK cells, respectively. NK cells were functional in terms of cytotoxicity, degranulation and antibody-dependent cell-mediated cytotoxicity.

Influenza A Virus Hemagglutinin and Other Pathogen Glycoprotein Interactions with NK Cell Natural Cytotoxicity Receptors NKp46, NKp44, and NKp30.

Natural killer (NK) cells are part of the innate immunity repertoire, and function in the recognition and destruction of tumorigenic and pathogen-infected cells. Engagement of NK cell activating receptors can lead to functional activation of NK cells, resulting in lysis of target cells. NK cell activating receptors specific for non-major histocompatibility complex ligands are NKp46, NKp44, NKp30, NKG2D, and CD16 (also known as FcγRIII). The natural cytotoxicity receptors (NCRs), NKp46, NKp44, and NKp30, have been implicated in functional activation of NK cells following influenza virus infection via binding with influenza virus hemagglutinin (HA). In this review we describe NK cell and influenza A virus biology, and the interactions of influenza A virus HA and other pathogen lectins with NK cell natural cytotoxicity receptors (NCRs). We review concepts which intersect viral immunology, traditional virology and glycobiology to provide insights into the interactions between influenza virus HA and the NCRs. Furthermore, we provide expert opinion on future directions that would provide insights into currently unanswered questions.

Activated Natural Killer Cells Are Associated with Poor Clinical Prognosis in High-Risk B- and T- Cell Acute Lymphoblastic Leukemia

Background: High-risk B- and T- cell Acute Lymphoblastic Leukemia (B/T-ALL) are aggressive lymphoid malignancies being treated with inhibitors targeting oncogenes and CAR-T cell-based immunotherapies. Despite advances in treatment, emergence of therapeutic resistance poses a clinical challenge in combating B/T-ALL. More recently, NK cells have emerged as attractive immunotherapies for cancers. Furthermore, NK therapies show promise for T-ALL, that has been particularly hard to treat with CAR-T cells. Approach: To develop NK therapies against B/T-ALL, it is important to identify the phenotypic and functional deficiencies in NK cells of ALL patients. Therefore, using mass cytometry (CyTOF), we compared the profile of NK cells in bone marrow (BM) or peripheral blood (PB) of 20 high-risk ALL patients (13 B-ALL, 7 T-ALL, 9 pediatric and 11 adult) and 22 tissue-matched healthy donors. We validated our CyTOF results by conducting in silico cytometry (CIBERSORT) for NK subsets in an independent panel of 207 high-risk B-ALL patients from the Children's Oncology Group (COG) P9906 clinical trial. We then investigated whether distinct NK signatures identified by CyTOF and CIBERSORT in B/T-ALL correlate with risk features and predict clinical outcome. Results: We observed a significant reduction in the frequency of total NK cells within the non-malignant immune fraction in B/T-ALL. Upon comparing the frequencies of specific NK subsets (CD56Brightand CD56Dim, we found that CD56Bright NK cells were increased in the BM (P=0.0138) but were reduced in PB (P=0.0285) of patients as compared to healthy donors. We then conducted CIBERSORT to estimate the relative proportions of NK cells with CD56Bright and CD56Dim molecular signatures in 207 COG B-ALL patients compared to 94 healthy donors. We found that frequencies of NK cells with CD56Bright molecular signature were increased in both BM and PB of ALL patients (P<0.0001). We conclude that majority of NK cells in B/T-ALL exhibit the molecular profile of CD56Bright NK cells, albeit their PB NK cells show reduced surface CD56. Human NK cells mature from CD56Bright to CD56Dim stages. CD56Dim NK are more cytotoxic and produce less cytokines than CD56Bright NK cells. Enrichment of CD56Bright molecular signature in NK cells of B/T-ALL patients suggested an impairment in NK maturation and function in ALL. By comparing the abilities of PB NK cells in B/T-ALL and healthy donors to lyse K562 targets invitro, we found that NK cells in ALL patients are poorly cytolytic compared to healthy donors (P=0.0295). Upon stimulation with PMA/Ionomycin, we observed an increase in the frequencies of GM-CSF+ and TNF-α+ cells in total NK and NK subsets in BM and PB of B/T-ALL patients compared to healthy donors (all P<0.05). Surprisingly, the natural cytotoxicity receptor NKp46 was increased in stimulated NK subsets of B/T-ALL patients compared to healthy controls (P<0.01). We conclude that poorly cytotoxic NK cells with CytokineHigh NKp46High activated phenotype (NKActiv) are enriched in B/T-ALL, while normal NK phenotype (NKRest) is suppressed. Finally, we investigated whether relative frequencies of NKActiv and NKRest cells can predict clinical outcome in high-risk ALL. To this end, we used CIBERSORT to estimate proportions of NK cells with activated (NKActiv) and resting (NKRest) molecular signatures in 207 COG B-ALL patients. We then separated patients into 2 groups as NKActiv> NKRest and NKRest> NKActiv, and compared their relapse-free survival (RFS) probabilities (Fig.1). We found that patients with higher NKActiv cells had shorter RFS than those with higher NKRest cells (P=0.0028). Furthermore, we found that relative proportions of activated and resting NK cells independently predict clinical outcome within a poorly prognostic subset of patients with Central Nervous System involvement (CNS+, P=0.0098). These preliminary findings suggest that higher levels of activated NK cells in B-ALL may be associated with poor clinical prognosis. We are validating these results in B/T-ALL from our CyTOF studies. Conclusion: We find that impairment of NK maturation in high-risk human B/T-ALL results in the accumulation of NK cells with a poorly cytotoxic but hyperactivated cytokine-producing phenotype, that positively correlates with poor clinical prognosis. Our data suggest that restoring NK homeostasis would be an attractive strategy for treating high-risk B/T-ALL. Disclosures Marcucci: Novartis: Speakers Bureau; Abbvie: Speakers Bureau; Iaso Bio: Membership on an entity's Board of Directors or advisory committees; Takeda: Other: Research Support (Investigation Initiated Clinical Trial); Merck: Other: Research Support (Investigation Initiated Clinical Trial); Pfizer: Other: Research Support (Investigation Initiated Clinical Trial).

The Antitumor Effect of Heparin is not Mediated by Direct NK Cell Activation.

Natural killer (NK) cells are innate lymphocytes responsible for the elimination of infected or transformed cells. The activation or inhibition of NK cells is determined by the balance of target cell ligand recognition by stimulatory and inhibitory receptors on their surface. Previous reports have suggested that the glycosaminoglycan heparin is a ligand for the natural cytotoxicity receptors NKp30, NKp44 (human), and NKp46 (both human and mouse). However, the effects of heparin on NK cell homeostasis and function remain unclear. Here, we show that heparin does not enhance NK cell proliferation or killing through NK cell activation. Alternatively, in mice models, heparin promoted NK cell survival in vitro and controlled B16-F10 melanoma metastasis development in vivo. In human NK cells, heparin promisingly increased interferon (IFN)-γ production in synergy with IL-12, although the mechanism remains elusive. Our data showed that heparin is not able to increase NK cell cytotoxicity.

Transcriptional Regulation of the Natural Cytotoxicity Receptor NKp44 Gene in Human NK Cell Leukemia

The natural cytotoxicity receptor NKp44 is an important regulator of NK cell function. We have previously reported that NKp44 has binding affinity to heparin and the transferrin containing multimeric sialyl Lewis X produced by human hepatoma HepG2 cells (HepTF) is more highly expressed than the other natural cytotoxicity receptors. To further understand the differences between natural cytotoxicity receptors, we sought to understand the gene transcriptional regulation of NKp44 in KHYG-1 human NK leukemia cells. 5′-rapid amplification of cDNA ends revealed the location of the NKp44 transcription initiation sites allowing identification of the 5′ untranscribed region. The result of the dual luciferase assay suggested that regulation of NKp44 gene expression is affected by the -1963 to -1599 nt region and the -352 to -231 nt region relative to the transcription initiation sites. The -410 to +1 nt region contained potential binding sites for the transcription factors AP-1, Oct-1, HNF-4, and Pax-4. These results suggest that defined regions in the 5′-flanking region of NKp44 gene is important for NKp44 transcription and regulation.

Distinct Human NK Cell Phenotypes and Functional Responses to Mycobacterium tuberculosis in Adults From TB Endemic and Non-endemic Regions.

Mycobacterium tuberculosis (Mtb) is the causative agent of tuberculosis (TB), which leads to an estimated 1. 5 million deaths worldwide each year. Although the immune correlates of protection against Mtb infection and TB disease have not been well-defined, natural killer (NK) cells are increasingly recognized as a key component of the innate immune response to Mtb and as a link between innate and adaptive immunity. In this study, we evaluated NK cell phenotypic and functional profiles in QuantiFERON-TB (QFT)+ and QFT− adults in a TB endemic setting in Kisumu, Kenya, and compared their NK cell responses to those of Mtb-naïve healthy adult controls in the U.S. We used flow cytometry to define the phenotypic profile of NK cells and identified distinct CD56dim NK cell phenotypes that differentiated the Kenyan and U.S. groups. Additionally, among Kenyan participants, NK cells from QFT+ individuals with latent Mtb infection (LTBI) were characterized by significant downregulation of the natural cytotoxicity receptor NKp46 and the inhibitory receptor TIGIT, compared with QFT− individuals. Moreover, the distinct CD56dim phenotypic profiles in Kenyan individuals correlated with dampened NK cell responses to tumor cells and diminished activation, degranulation, and cytokine production following stimulation with Mtb antigens, compared with Mtb-naïve U.S. healthy adult controls. Taken together, these data provide evidence that the phenotypic and functional profiles of NK cells are modified in TB endemic settings and will inform future studies aimed at defining NK cell-mediated immune correlates that may be protective against acquisition of Mtb infection and progression to TB disease.

Regulation of NK-Cell Function by HLA Class II

Natural Killer (NK) cells were initially described as part of the innate immune system and characterized by their ability to lyse malignant and virus-infected cells. The cytolytic function of NK cells is tightly controlled by activating and inhibitory receptors expressed on the cell surface. Ligands that interact with a variety of NK-cell receptors include the human leukocyte antigen (HLA) molecules, and the regulation of NK-cell function by HLA class I molecules is well-established. Earlier studies also suggested a role of HLA class II molecules in regulating NK cell activity; yet, interactions between HLA class II molecules and NK cell receptors have not been well-characterized. We recently identified a subset of HLA-DP molecules that can serve as ligands for the natural cytotoxicity receptor NKp44 and activate NK cells. This novel receptor-ligand interaction provides a potential mechanism to explain the strong associations of HLA-DP molecules with HBV infection outcomes, graft-vs.-host disease and inflammatory bowel disease. Furthermore, it adds a new mechanism for NK-cell crosstalk with immune cells expressing HLA class II molecules. In this perspective article, we discuss the potential implications of NK cell receptor interactions with HLA class II molecules for the regulation of immune responses.

Genome-Wide CRISPR Screen Reveals Cancer Cell Resistance to NK Cells Induced by NK-Derived IFN-γ.

The anti-leukemia activity of NK cells helps prevent relapse during hematopoietic stem cell transplantation (HSCT) in leukemia patients. However, the factors that determine the sensitivity or resistance of leukemia cells in the context of NK-mediated cytotoxicity are not well-established. Here, we performed a genome-wide CRISPR screen in the human chronic-myelogenous-leukemia (CML) cell line K562 to identify genes that regulate the vulnerability of leukemia cells to killing by primary human NK cells. The distribution of guide RNAs (gRNAs) in K562 cells that survived co-incubation with NK cells showed that loss of NCR3LG1, which encodes the ligand of the natural cytotoxicity receptor NKp30, protected K562 cells from killing. In contrast, loss of genes that regulate the antigen-presentation and interferon-γ-signaling pathways increased the vulnerability of K562 cells. The addition of IFN-γ neutralizing antibody increased the susceptibility of K562 cells to NK-mediated killing. Upregulation of MHC class I on K562 cells after co-incubation with NK cells was dependent on IFNGR2. Analysis of RNA-seq data from The Cancer Genome Atlas (TCGA) showed that low IFNGR2 expression in cancer tissues was associated with improved overall survival in acute myeloid leukemia (AML) and Kidney Renal Clear Cell Carcinoma (KIRC) patients. Our results, showing that the upregulation of MHC class I by NK-derived IFN-γ leads to resistance to NK cytotoxicity, suggest that targeting IFN-γ responses might be a promising approach to enhance NK cell anti-cancer efficacy.

Vδ1+ T Cells: Adoptive Cell Therapy for the Treatment of Haematological Malignancies in Allogeneic Settings

The treatment of haematological malignancies with adoptive cell therapy such as leukaemia is limited to conventional ab T cells transduced at great cost with chimeric antigen receptors (CAR) recognising the B-cell associated antigen CD19. Although greatly successful especially in paediatric cases, this therapy remains with its challenges such as associated toxicities, relapse rate as well as high production costs and production failure rates due to its autologous nature, indicated by the high risk of αβ T cells inducing sever graft versus host disease (GvHD). In contrast to αβ T cells, γδ T cells are not restricted to, or recognise major histocompatibility complex (MHC) peptide complexes. Especially Vδ1 T cells, enriched in human tissues but also found in small numbers in the human blood have been shown to elicit potent anti-tumour responses recognising stress ligands up-regulated on malignant cells, whilst sparing healthy tissue. A study looking at 39 malignancies, solid tumours as well as haematological malignancies, identified the presence of γδ T cells as the single most positive correlative factor for overall survival. Even more striking, expanding γδ T cells and more specifically Vδ1 T cells correlate with strong graft versus leukaemia and reduced incident rates of GvHD after αβ T cell depleted, haploidentical stem cell transplantation. These correlative findings have been confirmed in several retrospective data-sets but more recently in a prospective study looking at multiple haematological indications such as AML, ALL and MM. We have developed a 2½ week, good manufacturing practice (GMP) compliant and scalable (≥ 1x109) process to consistently generate αβ T cell depleted, γδ T cell lymphocyte cultures (93.5% ± 3.7 of live), strongly enriched for Vδ1 T cells (67.9%±2.2% of live). Vδ1 T cells show good recoverability and proliferation in response to cytokines after cryopreservation, kill a variety of malignant leukaemia and myeloma cells at low effector to target ratios (2:1) and show a favourable innate phenotype expressing high levels of the co-stimulatory molecule CD27, CD69 and the natural cytotoxicity receptors NKG2D, DNAM-1 and NKp30. Expression of negative modulators such as PD-1, CTLA4 and TIGIT remain low or non-detectable. Vδ1 T cells produce large levels of IFN-γ, TNF-α but little to no IL-17 on activation. Furthermore, large amounts of the pro-inflammatory chemokines CCL2 and CLL5, as well as the antigen-presenting cell attracting chemokine CCL4 additionally to the colony stimulating factors M-CSF and GM-CSF, support the idea of Vδ1 T cells orchestrating secondary adaptive immune responses after activation. Importantly, and in accordance with their MHC-independent biology, Vδ1 T cells do not kill healthy lymphocytes or primary tissue-derived stromal cells such as fibroblasts. Vδ1 T cells derived from healthy blood and generated under GMP conditions constitute a potential 'off-the-shelf', fully allogeneic cell therapy for the treatment of multiple haematological malignancies. Disclosures Simoes: GammaDelta Therapeutics Ltd.: Employment. Kawalkowska:GammaDelta Therapeutics Ltd.: Employment. Recaldin:GammaDelta Therapeutics Ltd.: Employment. Foxler:GammaDelta Therapeutics Ltd.: Employment. Nussbaumer:GammaDelta Therapeutics Ltd.: Employment, Equity Ownership, Patents & Royalties. Koslowski:GammaDelta Therapeutics Ltd.: Employment, Equity Ownership.

Human cytomegalovirus IL-10 augments NK cell cytotoxicity.

Human cytomegalovirus (HCMV) persistently infects most of the adult population with periods of productive and latent infection differentially orchestrated by multiple HCMV-encoded gene products. One HCMV gene (UL111a) encodes cmvIL-10, a virokine homologous to human IL (hIL)-10. Although the effects of cmvIL-10 on most human lymphocyte subsets have been extensively studied, its impact on NK cell function was unreported prior to this study. We investigated effects of short-term cmvIL-10 exposure on human NK cells and found it substantially enhanced NK cell cytotoxicity through natural cytotoxicity receptors NKp30 and NKp46 as well as through C-type lectin-like receptors NKG2C and NKG2D. Antibody-dependent cell-mediated cytotoxicity triggered through CD16 also increased significantly with short-term cmvIL-10 exposure. These effects of cmvIL-10 on NK cell cytotoxicity were rapid, dose dependent, neutralized by polyclonal anti-cmvIL-10 or monoclonal anti-IL-10 receptor (IL-10R) antibodies and independent of increased perforin synthesis or up-regulation of activating receptors. A low percentage (0.5-5.4%; n=12) of NK cells expressed IL-10R and the impact of cmvIL-10 on NK cells degranulation following CD16 stimulation directly correlated with this percentage (P= 0.0218). Short-term exposure of human NK cells to cmvIL-10 did not introduce phenotypic changes reminiscent of NK adaptation to HCMV infection in vivo. Determining how expression of a viral protein that activates NK cells contributes to their function in vivo will increase understanding of HCMV infection and NK cell biology.

Commensal and Pathogenic Bacteria Indirectly Induce IL-22 but Not IFNγ Production From Human Colonic ILC3s via Multiple Mechanisms.

Innate lymphoid cells (ILCs) are a diverse family of cells that play critical roles in mucosal immunity. One subset of the ILC family, Group 3 ILCs (ILC3s), has been shown to aid in gut homeostasis through the production of IL-22. IL-22 promotes gut homeostasis through its functional effect on the epithelial barrier. When gut epithelial barrier integrity is compromised, such as in Human Immunodeficiency Virus (HIV) infection and inflammatory bowel disease (IBD), microbes from the gut lumen translocate into the lamina propria, inducing a multitude of potentially pathogenic immune responses. In murine models of bacterial infection, there is evidence that bacteria can induce pro-inflammatory IFNγ production in ILC3s. However, the impact of diverse translocating bacteria, particularly commensal bacteria, in dictating IFNγ versus IL-22 production by human gut ILC3s remains unclear. Here, we utilized an in vitro human lamina propria mononuclear cell (LPMC) model to evaluate ILC3 cytokine production in response to a panel of enteric Gram-positive and Gram-negative commensal and pathogenic bacteria and determined potential mechanisms by which these cytokine responses were induced. The percentages of IL-22-producing ILC3s, but not IFNγ-producing ILC3s, were significantly increased after LPMC exposure to both Gram-positive and Gram-negative commensal or pathogenic bacterial stimuli. Stimulation of IL-22 production from ILC3s was not through direct recognition of bacterial antigen by ILC3s, but rather required the help of accessory cells within the LPMC population. CD11c+ myeloid dendritic cells generated IL-23 and IL-1β in response to enteric bacteria and contributed to ILC3 production of IL-22. Furthermore, ligation of the natural cytotoxicity receptor NKp44 on ILC3s in response to bacteria stimulation also significantly increased the percentage of IL-22-producing ILC3s. Overall, these data demonstrate that human gut microbiota, including commensal bacteria, indirectly modulate colonic ILC3 function to induce IL-22, but additional signals are likely required to induce IFNγ production by colonic ILC3s in the setting of inflammation and microbial translocation.

Deficient Natural Killer Cell NKp30-Mediated Function and Altered NCR3 Splice Variants in Hepatocellular Carcinoma.

The activating natural cytotoxicity receptor NKp30 is critical for natural killer (NK) cell function and tumor immune surveillance. The natural cytotoxicity receptor-3 (NCR3) gene is transcribed into several splice variants whose physiological relevance is still incompletely understood. In this study, we investigated the role of NKp30 and its major ligand B7 homolog 6 (B7-H6) in patients with hepatocellular carcinoma (HCC). Peripheral blood NK cell phenotype was skewed toward a defective/exhausted immune profile with decreased frequencies of cells expressing NKp30 and natural killer group 2, member D and an increased proportion of cells expressing T-cell immunoglobulin and mucin-domain containing-3. Moreover, NKp30-positive NK cells had a reduced expression of NCR3 immunostimulatory splice variants and an increased expression of the inhibitory variant in patients with advanced tumor, resulting in deficient NKp30-mediated functionality. Tumor-infiltrating lymphocytes showed a prevalent inhibitory NKp30 isoform profile, consistent with decreased NKp30-mediated function. Of note, there were significant differences in the cytokine milieu between the neoplastic and the surrounding non-neoplastic tissue, which may have further influenced NKp30 function. Exposure of NK cells to B7-H6-expressing HCC cells significantly down-modulated NKp30, that was prevented by small interfering RNA-mediated knockdown, suggesting a role for this ligand in inhibiting NKp30-mediated responses. Interestingly, B7-H6 expression was reduced in HCC tissue and simultaneously augmented as a soluble form in HCC patients, particularly those with advanced staging or larger nodule size. Conclusion: These findings provide evidence in support of a role of NKp30 and its major ligand in HCC development and evolution.

Type 3 innate lymphoid cells are altered in colons of C57BL/6 mice with dioxin exposure

Type 3 innate lymphoid cells (ILC3s) are distributed in the gut and regulate inflammation by secreting cytokines, including interferon (IFN)-γ and interleukin (IL)-17. The maintenance and function of ILC3s involve the activity of aryl hydrocarbon receptor (AhR), a potent ligand of which is 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), one of the most toxic dioxin congeners. Thus, TCDD exposure might affect ILC3s. To obtain in vivo evidence supporting this notion, we exposed female C57BL/6 mice orally to TCDD (low/high doses: 0.1/10 μg/kg body weight) during pregnancy and lactation periods, and after the exposure, evaluated the mothers and offspring for alterations in ILC3 differentiation and function in the colon. ILC3 frequency among colonic lamina propria lymphocytes was preferentially diminished in the offspring, and, in parallel, the median fluorescence intensity (MFI) of retinoic acid receptor-related orphan receptor (ROR)γt, which is associated with ILC3 differentiation, was also decreased in ILC3s. Conversely, the percentages of two subsets of the cells, one positive for natural cytotoxicity receptor NKp46 and the other for IL-17a, were increased in TCDD-exposed mothers and offspring. Moreover, the percentage of IFN-γ+ ILC3s was increased specifically in the mothers, but this was in conjunction with a significant decrease in the MFI of IFN-γ, which suggests that the IFN-γ+ ILC3 subset was functionally altered. In conclusion, maternal exposure to TCDD suppresses ILC3 differentiation in the offspring and influences ILC3 function in distinct manners in the mother and offspring. Our study provides new insights into the intergenerational interference of dioxins in colonic ILC3s.

Globo-A Binds to the Recombinant Natural Cytotoxicity Receptor NKp44.

Natural killer (NK) cells play an important role in tumor immunity and infection control. The natural cytotoxicity receptors (NCRs) NKp46, NKp44 and NKp30 are involved in the control of the activation of NK cells. Few reports have investigated the ligands of NCRs. We previously reported the NCRs binding affinity to heparin and glycosaminoglycans. We also showed that multimeric sialyl Lewis X-expressing transferrin, secreted by human hepatoma HepG2 cells, binds to NKp46 and NKp44, but not to NKp30. In this study, we investigated the binding between NCRs and glycolipids. The possible binding of glycolipids to NCRs was screened by microarray, using the recombinant extracellular domain of NKp46, NKp44 and NKp30 tagged with 6×His (rNKp46, rNKp44 and rNKp30). We found that rNKp44 binds to Globo-A. However, we did not detect the interaction between rNKp46 or rNKp30 and any of the glycolipids investigated. Direct binding assays supported the results of the microarray screening. Therefore, we concluded that Globo-A is a novel ligand for NKp44 but not NKp46 and NKp30, and showed differences in the ligand selectivity of NCRs.