Inhibitory Natural Killer Receptors Research Articles

Cell therapy using anti-NKG2A pretreated natural killer cells in patients with hepatocellular carcinoma

Background: The activities and functions of natural killer (NK) cells are controlled by a limited repertoire of activating and inhibitory NK receptors. Therefore, blocking inhibitory receptors such as NKG2A using monoclonal antibodies (mAbs) enhances tumor immunity. Method: In this study, we investigated the safety of anti-NKG2A-pretreated NK cells in improving ADCC function to manage hepatocyte carcinoma (HCC). After a conditioning regimen, we initiated a pilot study of expanded donor haploidentical NK cell infusion. The goals were to determine the safety and feasibility. Patients received a fludarabine/cyclophosphamide conditioning followed by adoptive immunotherapy with IL2–activated haploidentical NK cells. Anti-NKG2A pretreated NK cells were infused on days 0, +5, and +10 post-conditioning regimens at a dose of 7 × 108 cells (n=3). The median follow-up was 4 months for all patients. Results: Although all patients were alive at the last follow-up, two of them showed progressive disease and an increase in tumor size. In addition, all patients had a relative decrease in the expression level of an alpha-fetoprotein (AFP) after one month. Conclusion: This study demonstrated the safety and feasibility of infusing high doses of ex vivo expanded NK cells after conditioning with transient side effects.

Abstract C020: Discovery & characterization of small molecules to enhance Natural Killer cell cytotoxicity against solid tumors

Abstract While adoptive cellular therapies (ACT) such as CAR-T and NK therapy have shown clinical efficacy against hematologic malignancies, their use against solid tumors (such as ovarian or pancreatic cancer) has had limited success to date. In particular, CAR-T therapy for solid cancers has led to severe toxicities, including on-target, off-tumor killing of healthy cells and graft vs. host disease (GVHD) with allogeneic transplant. NK cell therapies, in contrast, have fewer associated toxicities due to specific and innate recognition/killing of cancer cells. This greater safety profile makes NK therapies an attractive treatment option for solid cancers. NK therapies are currently limited by insufficient in vivo tumor clearance, caused largely by their lower cytotoxicity and in vivo expansion compared to CAR-T cells. To address this, we have screened 300,000 small molecule compounds for their ability to enhance NK cytotoxicity against ovarian cancer (OVCAR) cells. Our primary screen identified 11 lead compounds which increase NK killing activity against OVCAR cells >= 10% over baseline (vehicle-treated control). Secondary in vitro assays have confirmed that at least 3 out of 11 lead compounds enhance NK cytotoxicity by at least 10% over baseline. As such, we hypothesize that our lead compounds will increase in vivo NK cytotoxicity and tumor clearance, through mechanisms which might include I) upregulation of NK activating receptors, II) downregulation of NK inhibitory receptors, and/or III) modulation of NK metabolism in the immunosuppressive tumor microenvironment. Immediate future studies include in vivo testing of our lead compounds’ ability to enhance NK cytotoxicity, using immunodeficient mouse models of subcutaneous OVCAR tumors. Moreover, we will utilize 2D and 3D in vitro culture techniques alongside flow cytometry and RNA sequencing to identify how lead compounds impact markers of NK activation and metabolism. This will include expression of activating/inhibitory receptors, exhaustion markers, and key metabolic factors. Finally, we will assess the effect of lead compounds on NK function within an immunosuppressive tumor environment. Our findings will contribute to effective future NK cell therapies for solid tumors. Citation Format: Indrani Das, Olubukola Abiona, Riufu Liu, Grace K. Lee, David N. Wald. Discovery & characterization of small molecules to enhance Natural Killer cell cytotoxicity against solid tumors [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor-body Interactions: The Roles of Micro- and Macroenvironment in Cancer; 2024 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(22_Suppl):Abstract nr C020.

NKG2C/KLRC2 tumor cell expression enhances immunotherapeutic efficacy against glioblastoma

BackgroundActivating and inhibitory receptors of natural killer (NK) cells such as NKp, NKG2, or CLEC are highly relevant to cold tumors including glioblastoma (GBM). Here, we aimed to characterize the...

P162 Dimethyl fumarate modulates T cell metabolism and function in systemic lupus erythematosus patient samples

Abstract Background/Aims Activated CD4+T cells make a significant contribution to inflammation in systemic lupus erythematosus (SLE) through secretion of proinflammatory cytokines such as IFN-γ and TNF-α. CD4+T cells can also express activating NK receptors such as NKG2D that can engage with stress-induced ligands such as MICA, as noted in lupus nephritis, and mediate nonspecific tissue injury. We know that cellular metabolism regulates the activation of T cells. Evidence from patients with multiple sclerosis indicates that dimethyl fumarate (DMF), an electrophile, targets cellular metabolism to modulate CD4+T cell activation and function. Here, we investigated whether DMF modulates T cell metabolism and function in samples from patients with SLE in a series of in vitro experiments. Methods All experiments were performed using isolated T cells from freshly drawn whole blood samples from patients with SLE. T cells were isolated using negative selection using stem cell or Miltenyei magnetic bead separation kit. Isolated T cells were activated with anti-CD3 and IL-2 and incubated with either DMF at 25μM concentration or DMSO alone for three or seven days at 37 °C and 5% CO2 before harvesting. Results In Seahorse experiments, after three days of incubation, dimethyl fumarate (DMF) inhibited Figure A-i) the oxygen consumption rate (OCR) and A-ii) extra cellular acidification rate (ECAR) in isolated T cells when compared with samples incubated with vehicle, dimethyl sulfoxide (DMSO). Our results revealed that DMF significantly inhibited: 1) aerobic glycolysis and oxidative phosphorylation in activated T cells from patients with SLE (n = 4) (Figure A- i and ii) in vitro; 2) T cell activation and proliferation as assessed by a reduction in the frequency of CD69 (n = 4) and D) Ki67 (n = 2) positivity, respectively (Figure B-E); 3) Collectively, these results suggest that DMF inhibits T cell activation and proliferation in samples from patients with SLE. After 7 days of incubation, DMF significantly inhibited the expression of activating NK receptors CD158a and NKG2D on T cells whereas there was a trend toward enhancing the expression of inhibitory NK receptors NKG2A and CD158b (Figure F-I). After 7 days of incubation, DMF significantly reduced intracellular expression of IFN-γ, TNF-α, IL-17 and secretion of pro-inflammatory cytokines IFN-γ and TNF-α in supernatants (Figure J - N). Conclusion Our preliminary data indicated that, in SLE patient samples, DMF modulates metabolic programming of CD4+ T cells and inhibits glycolysis and oxidative phosphorylation of activated CD4+ T cells. In vitro, DMF inhibited: T cell activation; proliferation; the expression of activating NK receptors; and secretion of proinflammatory cytokines from T cells patients with SLE. Taken together with the existing safety data in humans, these data provide mechanistic rationale for investigating the potential of dimethyl fumarate, with a distinct mechanism of action, as a novel therapy for SLE. Disclosure L. Kell: None. K. Shah: None. S. Taylor: None. R. De Maeyer: None. D.A. Isenberg: None. D. Sen: None. M. Castelino: None. M. Leandro: None. A. Akbar: None. V. Reddy: Grants/research support; UCLH Biomedical research centre funded this work, Dr.Reddy's work was supported through MR/T024968/1 MRC-CARP Fellowship.

Abstract 2687: NK cell plasticity in SCLC is linked to tumor transcriptional diversity

Abstract Natural Killer (NK) cells are innate lymphoid cells that are pivotal in the body’s response to cancer. To harness their cytotoxicity in clinical interventions, it is critical to understand their biology, which is highly tissue- and tumor-specific. In Small Cell Lung Cancer (SCLC), reports have described widely divergent levels of NK activity in neuroendocrine (NE) and non-neuroendocrine (non-NE) subtypes. Diminished self-antigen presentation by MHC-I is a classic trigger of NK cell activity but, counter-intuitively, NK cells exhibit reduced cytotoxicity in MHC-I downregulated NE SCLC. To explore potential causes for depressed NK activity in NE SCLC relative to non-NE, we applied scRNA-Seq to 45 SCLC patient biopsies and rapid autopsies, and identified 4000 NK cells based on gene expression signature. There was a notable dip in NK cell infiltration in NE tumors compared to non-NE tumors. Tumor cell-surface ligands recognized by activating and, unexpectedly, inhibitory NK cell receptors were generally both more highly expressed in non-NE tumors. While inhibitory NK receptors were comparable in NE and non-NE tumors, activating NK receptors were expressed significantly, though modestly, higher in non-NE. We observed tumor site-specific gene expression patterns in NK cells from different metastatic sites. Overall, there were a remarkably high number of differentially expressed genes (>1k) between NK cells from NE and non-NE tumors, even after controlling for tumor site. Together, our work suggests that NK cell plasticity is manifest at the level of SCLC transcriptional subtypes. Citation Format: Justin Malin, Sophie Zhuang, Ajit Kumar Sharma, Joseph Clara, Anish Thomas. NK cell plasticity in SCLC is linked to tumor transcriptional diversity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2687.

Abstract 179: Identification of a novel crosstalk between MDSC and NKregs in immunotherapy resistant triple negative breast cancer

Abstract Triple-negative breast cancer (TNBC) is one of the most aggressive forms of breast cancer and lacks standard treatment options. Although immunotherapy shows some response in these patients along with chemotherapy, heterogeneity of tumor cells and immune cells results in significant low response to immunotherapy treatment. Based on single cell sequencing which enables identity of heterogenous population at single cell level and functional studies involving preclinical mouse models, our laboratory recently identified distinct CD56brightSOCS3highCD11b−CD27− immature NK cells subpopulations, with diminished cytotoxic granzyme signatures that promote TNBC tumor progression. However, the precise characterization and functionality of these rogue tumor promoting NK cells remains unclear in clinical samples. Here, we aimed to illustrate mechanistic insight of these novel immature NK subpopulation in context of immunotherapy resistant TNBC patient samples. The scRNA seq analysis of TNBC patient samples identified four specific subclusters of NK cells in TNBC. We further found that NK2 and NK3 subclusters are CD56dim Granzymehigh cytotoxic NK cells and the other two subclusters (NK0 and NK1) are CD56bright immature NKs. Interestingly, the NK0 and NK1 subcluster specifically exhibit distinctive characteristics and functions. Notably, CD56bright immature NK1 cells, identified as tissue-resident NK cells, were engaged in direct communication with myeloid-derived suppressor cells (MDSCs). The CD56bright NK0 possesses moderate to low SOCS3 expression and moderate to high granzyme expression with elevated early NK activation markers and inhibitory receptor expressions, suggesting its regulatory property (NKregs). scRNA seq and flow cytometry analysis showed higher NKregs and immunosuppressive MDSC population, specifically with high monocytic-MDSCs (mMDSCs) in non-responder TNBC (to adjuvant combinational treatment of chemo and immunotherapy) patient samples rather than responder patients’ tissue. Based on in silico analysis, we further found a potential crosstalk between these mMDSC & NKreg within the tumors of non-responsiveness of TNBC patients. Overall, our studies show a connection of MDSCs and NKregs in TNBC patients, who do not respond to immunotherapy, highlighting a possible mechanism for immunotherapy resistance, which could be targeted in near future for better treatment options in TNBC. Citation Format: Megha Das, Samantha Henry, Shailesh Singh, Camila Dos Santos, Rumela Chakrabarti. Identification of a novel crosstalk between MDSC and NKregs in immunotherapy resistant triple negative breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 179.

BET inhibitors drive Natural Killer activation in non-small cell lung cancer via BRD4 and SMAD3

Non-small-cell lung carcinoma (NSCLC) is the most common lung cancer and one of the pioneer tumors in which immunotherapy has radically changed patients’ outcomes. However, several issues are emerging and their implementation is required to optimize immunotherapy-based protocols. In this work, we investigate the ability of the Bromodomain and Extra-Terminal protein inhibitors (BETi) to stimulate a proficient anti-tumor immune response toward NSCLC. By using in vitro, ex-vivo, and in vivo models, we demonstrate that these epigenetic drugs specifically enhance Natural Killer (NK) cell cytotoxicity. BETi down-regulate a large set of NK inhibitory receptors, including several immune checkpoints (ICs), that are direct targets of the transcriptional cooperation between the BET protein BRD4 and the transcription factor SMAD3. Overall, BETi orchestrate an epigenetic reprogramming that leads to increased recognition of tumor cells and the killing ability of NK cells. Our results unveil the opportunity to exploit and repurpose these drugs in combination with immunotherapy.

Establishing a Pathogenic Role for NK Cells As Drivers of Bone Marrow Failure in the Myelodysplastic Syndromes (MDS)

Background: Myelodysplastic syndromes (MDS) are characterized by aberrant bone-marrow (BM) morphology leading to a clonal impairment of hematopoiesis. Worsening BM failure (BMF) and increasing myeloblasts are observed as the predominant consequences of MDS disease progression from very low risk (VLR) to higher risk (HR) to acute myeloid leukemia (AML). Patients are stratified based on the degree of BMF and risk of AML transformation to help guide treatment decisions. Approximately 30% of MDS patients exhibit various autoimmune diseases that can include autoantibodies targeting neutrophils, erythrocytes, and/or platelets. Autoimmune phenomena play an important role in MDS yet are poorly characterized. We hypothesized that early in LR-MDS, dysregulated interactions between stromal cells and developing Natural Killer (NK)/T-cells promote rapid/unregulated development resulting in autoreactivity ( Fig. 1A). We predict dual effector mechanisms: CD16-mediated antibody-dependent cellular cytotoxicity (ADCC) and loss of HLA class I (HLA-I) expression by developing hematopoietic lineage cells making them targets for “missing-self” reactivity ( Fig. 1A). NK cells are important sentinel cells that recognize tumors and dysfunctional cells that lose expression of HLA class I. Such surveillance is guided by interactions between NK inhibitory receptors and HLA class I ligands that ‘train’ NK cells to distinguish diseased cells, exhibiting perturbed HLA expression, from healthy cells with normal expression. The effects of these interactions combined with expression of activating receptors (e.g. CD16, NKG2D, DNAM1, FasL, TRAIL) allow NK cells to engage a target cell through many activating mechanisms, importantly including antibodies eliciting an antibody-dependent cellular cytotoxicity (ADCC) reaction. Autoantibodies are well-established in MDS, yet no one has profiled whether they may trigger ADCC/complement. Further, no one has ever thoroughly characterized BMMCs from LR-MDS patients before. In our study, we observed a clear link between signatures of bone marrow failure at LR-MDS and a significant increase in BM CD57+NK and CD8 T cells expressing a highly activated and dysregulated phenotype. Method: Ex-vivo profiling of 19 MDS patients (N=10 LR/IR-MDS, N=9 HR-MDS) BM mononuclear cells (BMMCs) was performed by mass cytometry (CyTOF)( Fig.1B). Cells were stained with three different antibody panels each consisting of 44 antibodies focused on: specific markers of hematopoiesis and myeloid or lymphoid cell lineages; specific markers of development, effector function, homing, exhaustion, and T-cell memory and proliferation; expression of HLA class I and activating ligands which are critical regulators of NK inhibition. Results: In our study we observed that a major fraction of MDS patients present with clonal-like expansions of BM-derived CD57+CD8+T-cells and natural killer (NK) cells that strongly correlate with autoimmune-cytopenias and BMF-enriched in LR-MDS (Fig.#). We observed that NK-cells and non-classical T-cells were significantly expanded (>2-fold) in LR MDS compared to HR-MDS and healthy-control BM (p<0.01). These non-classical CD57+CD8+T-cells co-expressed HLA-I-reactive and inhibitory NKG2A/KIR receptors as well as activating CD16, NKG2C, NKG2D, NKp46, TRAIL, FasL. In cancers, these cells use innate-like, TCR-independent mechanisms (traditionally ascribed to NK-cells) to target HLA-I-deficient tumor cells. Strikingly, HLA-I was significantly elevated on myeloblasts in LR-MDS compared to HR-MDS (p=0.0007)( Fig.1B), demonstrating NK-evasion strategies, but strongly reduced on dendritic cells, granulocytes, and CD4+T-cells, suggesting susceptible immune targets for destruction by NK-cells (and innate-like CD8+T-cells) (p< 0.0001) ( Fig.1B). BM-derived CD57+NK and CD8+T-cells all co-express very high levels of CD16 making them particularly ADCC reactive. Conclusion: We identified novel innate-like CD8+CD57+ T-cell and NK-cell populations that may play a role in LR-MDS disease pathogenesis. We predict that a strategy to target these cells could help restore hematopoiesis early in the disease state. This may also reduce pressure on myeloblasts to increase HLA-I, permitting improved immune surveillance to control the blast burden and prevention MDS/AML progression. Further experiments are ongoing to validate our observations.

Selinexor Enhances Natural Killer Cell Function Against Multiple Myeloma Cells

Introduction: Natural killer (NK) cells are innate immune effector cells which can mediate antibody dependent cellular cytotoxicity (ADCC) and lyse cancer cells. Harnessing NK cell function against multiple myeloma (MM) is of high interest and multiple strategies are currently under development. Exportin-1 (XPO1) is a nuclear export protein which inhibits the function of tumour suppressor proteins and facilitates oncogene mRNA translation. Selinexor is a first-in-class XPO1 inhibitor approved for the treatment of relapsed or refractory MM that induces cancer cell apoptosis. In addition, selinexor has recently been shown to promote NK cell cytotoxicity against B cell lymphoma cells. This study aimed to determine whether selinexor augments NK cell activation against MM cells both alone, and in combination with the anti-CD38 antibody daratumumab. Methods: A panel of MM cell lines (MM.1S, U266 and RPMI-8226) were incubated with clinically relevant concentrations of selinexor for 24 hours, then either assessed for expression of ligands for NK cell receptors (KIR, NKG2A, NKG2D and NKp46), or co-cultured with primary human NK cells ± daratumumab. Following co-culture, NK-mediated lysis of MM cells was assessed by propidium iodide staining and NK cell degranulation assessed by CD107a expression. Results: Selinexor pre-treatment of MM cells significantly increased NK cell degranulation (p<0.05) and enhanced NK specific lysis of MM targets (p<0.05). Furthermore, selinexor pre-treatment significantly (p<0.05) enhanced NK cell activation in combination with daratumumab compared to daratumumab alone. Importantly, selinexor did not alter surface expression of CD38 on MM cell lines. To investigate the mechanism behind this enhanced NK cell function, we assessed the effect of selinexor on the expression of NK cell ligands by MM cells. Selinexor increased the expression of ULBP1/2/5/6 (2-fold, p<0.001) which bind to the NK cell activating receptor NKG2D. In addition, selinexor decreased MM cell expression of HLA-E (30% decrease, p<0.05) which binds to the inhibitory NK receptor NKG2A. Conclusions: These data suggest that selinexor may promote the efficacy of NK targeted therapeutics in multiple myeloma.

Targeted Integration of a Chimeric Antigen Receptor in Key Selected Loci in Primary Natural Killer Cells

Despite promising preclinical studies and early clinical data (Liu et al., NEJM, 2020), chimeric antigen receptor natural killer cells (CAR NK) are not yet on par with CAR T cells for the treatment of malignant diseases. Much effort has gone into developing NK-optimized CAR architectures (Li et al., Cell Stem Cell, 2018) and to increasing the persistence of CAR NK cells. We hypothesized that CAR NK cells could also benefit from the targeted integration of a CAR transgene, as compared with semi-random integration obtained using gammaretroviral vectors. Precise targeting has proven highly advantageous for TRAC CAR T cells owing to the optimal regulation and homogeneous expression conferred by the endogenous TCR alpha promoter (Eyquem et al., Nature, 2017). We first optimized an editing protocol for primary NK cells that uses electroporation of a CRISPR/Cas9 ribonucleoprotein followed by transduction with AAV6 bearing a homology-directed repair template. With this protocol we were able to knock out (KO) genes (including CD38, B2M, and different NK receptors), knock in transgenes (GFP-fusion protein, truncated EGFR, CAR), or both, at high efficiency. There was only moderate cell toxicity, and proliferation and cytotoxicity were conserved. Because of the high constitutive expression of B2M, we initially chose to focus on this locus and targeted a 1928z CAR transgene for regulation under the endogenous B2M promoter; our template design also rescues the B2M gene upon knock-in, enabling intermediate B2M expression (Figure 1). This strategy conferred high expression of the CAR and significantly more homogeneity as compared with gammaretroviral delivery (Figure 1). Furthermore, the rescued B2M expression protected B2M CAR NK cells from fratricide killing, whereas the B2M KO NK cells and the CAR NK cells with disrupted B2M expression were susceptible. B2M CAR NK cells also displayed higher in vitro cytotoxicity as compared with WT NK cells against two different CD19-expressing tumor cell lines (NALM6, SupB15). We tested B2M CAR NK cells in an NSG mouse xenograft model with an intraperitoneally injected human ovarian cancer cell line engineered to express CD19 (CD19 + SKOV3). Unexpectedly, compared to retroviral CAR-NK cells, B2M CAR NK cells conferred less tumor control (assessed by bioluminescence imaging) and less of a survival benefit. Therefore, we screened a panel of other loci for CAR targeting: five for NK activating receptors ( CD16A, CD56, DNAM1, NKp30, NKp46) and four for NK inhibitory receptors ( CD161, LILRB1, NKG2A, TIGIT). For activating receptors, the transgene was designed to use the endogenous promoter and to maintain expression of the targeted locus. For inhibitory receptors, the transgene was placed under the control of an EF1a exogenous promoter and designed to disrupt expression of the targeted locus. We compared these constructs and the initial B2M CAR NK design for CAR expression (by flow cytometry) and in vitro functionality (cytotoxicity assays) and observed wide variation in knock-in efficiency and CAR expression (Figure 2). For the top constructs based on knock-in efficiency, we conducted in vitro cytotoxicity assays against NALM6 and SupB15 cell lines. We found that when normalized for CAR percentage using the respective KO conditions, all tested knock-in loci performed better than B2M CAR NK cells, despite the lower CAR expression. Our current focus is on validating these top loci for the targeted integration of a 1928z CAR using in vivo models for comparison with B2M CAR NK and retroviral CAR NK. In summary, we have identified advantageous in vitro characteristics for the targeted integration of a CAR transgene in primary NK cells and screened 10 loci for CAR expression and in vitro cytotoxicity against tumor target cells. Confirmatory in vivo studies are ongoing and will be presented.

MAb14, a Monoclonal Antibody against Cell Surface PCNA: A Potential Tool for Sezary Syndrome Diagnosis and Targeted Immunotherapy.

Mycosis fungoides (MF) and Sézary syndrome (SS) are the most common types of primary cutaneous T-cell lymphoma (CTCL). Proliferating cell nuclear antigen (PCNA) is expressed on the cell surface of cancer cells (csPCNA), but not on normal cells. It functions as an immune checkpoint ligand by interacting with natural killer (NK) cells through the NK inhibitory receptor NKp44, leading to the inhibition of NK cytotoxicity. A monoclonal antibody (mAb14) was established to detect csPCNA on cancer cells and block their interaction with NKp44. In this study, three CTCL cell lines and peripheral blood mononuclear cells (PBMCs) from patients with SS and healthy donors were analyzed for csPCNA using mAb14, compared to monoclonal antibody PC10, against nuclear PCNA (nPCNA). The following assays were used: immunostaining, imaging flow cytometry, flow cytometry, cell sorting, cell cycle analysis, ELISA, and the NK-cell cytotoxic assay. mAb14 successfully detected PCNA on the membrane and in the cytoplasm of viable CTCL cell lines associated with the G2/M phase. In the Sézary PBMCs, csPCNA was expressed on lymphoma cells that had an atypical morphology and not on normal cells. Furthermore, it was not expressed on PBMCs from healthy donors. In the co-culture of peripheral blood NK (pNK) cells with CTCL lines, mAb14 increased the secretion of IFN-γ, indicating the reactivation of pNK activity. However, mAb14 did not enhance the cytotoxic activity of pNK cells against CTCL cell lines. The unique expression of csPCNA detected by mAb14 suggests that csPCNA and mAb14 may serve as a potential biomarker and tool, respectively, for detecting malignant cells in SS and possibly other CTCL variants.

HLA-B*44 and the Bw4-80T motif are associated with poor outcome of relapse-preventive immunotherapy in acute myeloid leukemia.

HLA-B alleles are associated with outcomes in various pathologies, including autoimmune diseases and malignancies. The encodedHLA-B proteins are pivotal in antigen presentation to cytotoxic T cells, and some variants containing a Bw4 motif also serve as ligands to the killer immunoglobulin-like receptors (KIR) 3DL1/S1 of NK cells. We investigated the potential impact of HLA-B genotypes on the efficacy of immunotherapy for relapse prevention in acute myeloid leukemia (AML). Seventy-eight non-transplanted AML patients receiving HDC/IL-2 in the post-consolidation phase were genotyped for HLA-B and KIR genes. HLA-B*44 heralded impaired LFS (leukemia-free survival) and overall survival (OS), but the negative association with outcome was not shared across alleles of the HLA-B44 supertype. Notably, HLA-B*44 is one of few HLA-B44 supertype alleles containing a Bw4 motif with a threonine at position 80, which typically results in weak binding to the inhibitory NK receptor, KIR3DL1. Accordingly, a strong interaction between KIR3DL1 and Bw4 was associated with superior LFS and OS (p = 0.014 and p = 0.027, respectively). KIR3DL1+ NK cells from 80T-Bw4 donors showed significantly lower degranulation responses and cytokine responses than NK cells from 80I-Bw4 donors, suggesting impaired KIR3DL1-mediated education in 80T-Bw4 subjects. We propose that presence of a strong KIR3DL1+-Bw4 interaction improves NK cell education and thus is advantageous in AML patients receiving HDC/IL-2 immunotherapy for relapse prevention.

An efficient feeder-free and chemically-defined expansion strategy for highly purified natural killer cells derived from human cord blood

IntroductionNatural killer cells (NKCs) are immune cells that can attack cancer cells through the direct recognition of ligands without prior sensitization. Cord blood-derived NKCs (CBNKCs) represent a promising tool for allogenic NKC-based cancer immunotherapy. Efficient NKC expansion and decreased T cell inclusion are crucial for the success of allogeneic NKC-based immunotherapy without inducing graft-versus-host reactions. We previously established an efficient ex vivo expansion system consisting of highly purified-NKCs derived from human peripheral blood. Herein, we evaluated the performance of the NKC expansion system using CB and characterized the expanded populations. MethodsFrozen CB mononuclear cells (CBMCs), with T cells removed, were cultured with recombinant human interleukin (rhIL)-18 and rhIL-2 under conditions where anti-NKp46 and anti-CD16 antibodies were immobilized. Following 7, 14, and 21 days of expansion, the purity, fold-expansion rates of NKCs, and the expression levels of NK activating and inhibitory receptors were assessed. The ability of these NKCs to inhibit the growth of T98G, a glioblastoma (GBM) cell line sensitive to NK activity, was also examined. ResultsAll expanded T cell-depleted CBMCs were included in over 80%, 98%, and 99% of CD3−CD56+ NKCs at 7, 14, and 21 days of expansion, respectively. The NK activating receptors LFA-1, NKG2D, DNAM-1, NKp30, NKp44, NKp46, FcγRIII and NK inhibitory receptors TIM-3, TIGIT, TACTILE, NKG2A were expressed on the expanded-CBNKCs. Two out of three of the expanded-CBNKCs weakly expressed PD-1, yet gradually expressed PD-1 according to expansion period. One of the three expanded CBNKCs almost lacked PD-1 expression during the expansion period. LAG-3 expression was variable among donors, and no consistent changes were identified during the expansion period. All of the expanded CBNKCs elicited distinct cytotoxicity-mediated growth inhibition on T98G cells. The level of cytotoxicity was gradually decreased based on the prolonged expansion period. ConclusionsOur established feeder-free expansion system yielded large scale highly purified and cytotoxic NKCs derived from human CB. The system provides a stable supply of clinical grade off-the-shelf NKCs and may be feasible for allogeneic NKC-based immunotherapy for cancers, including GBM.

Combined flow cytometry natural killer immunophenotyping and KIR/HLA-C genotyping reveal remarkable differences in acute myeloid leukemia patients, but suggest an overall impairment of the natural killer response.

Natural killer (NK) cells are key anti-tumor effectors of the innate immunity. Phenotypic differences allow us to discriminate in between three functional stages of maturation, named immature, mature and hypermature that are distinctive in terms of receptor expression, cytokine secretion, cytotoxic properties and organ trafficking. NKs display an impressive repertoire of highly polymorphic germline encoded receptors that can be either activating, triggering the effector's function, or inhibitory, limiting the immune response. In our study, we have investigated peripheral blood NK cells of acute myeloid leukemia (AML) patients. The Killer Immunoglobulin-like receptors (KIRs) and the HLA-C genotypes were assessed, as HLA-C molecules are cognate antigens for inhibitory KIRs. The AA mainly inhibitory KIR haplotype was found in a higher proportion in AML, while a striking low frequency of the 2DS3 characterized the mainly activating Bx haplotype. Flow cytometry immunophenotyping evidenced a lower overall count of NK cells in AML versus healthy controls, with lower percentages of the immature and mature subpopulations, but with a markedly increase of the hypermature NKs. The analysis of the KIR2DL1, KIR2DL2, KIR2DL3, KIR3DL1, and NKG2A inhibitory receptors surface expression revealed a remarkable heterogeneity. However, an overall trend for a higher expression in AML patients could be noticed in all maturation subpopulations. Some of the AML patients with complex karyotypes or displaying a FLT3 gene mutation proved to be extreme outliers in terms of NK cells percentages or inhibitory receptors expression. We conclude that while the genetic background investigation in AML offers important pieces of information regarding susceptibility to disease or prognosis, it is flow cytometry that is able to offer details of finesse in terms of NK numbers and phenotypes, necessary for an adequate individual evaluation of these patients.

Human CD8+ T-Cell Populations That Express Natural Killer Receptors.

CD8+ T cells are activated by TCRs that recognize specific cognate Ags, while NK-cell activation is regulated by a balance between signals from germline-encoded activating and inhibitory NK receptors. Through these different processes of Ag recognition, CD8+ T cells and NK cells play distinct roles as adaptive and innate immune cells, respectively. However, some human CD8+ T cells have been found to express activating or inhibitory NK receptors. CD8+ T-cell populations expressing NK receptors straddle the innate-adaptive boundary with their innate-like features. Recent breakthrough technical advances in multi-omics analysis have enabled elucidation of the unique immunologic characteristics of these populations. However, studies have not yet fully clarified the heterogeneity and immunological characteristics of each CD8+ T-cell population expressing NK receptors. Here we aimed to review the current knowledge of various CD8+ T-cell populations expressing NK receptors, and to pave the way for delineating the landscape and identifying the various roles of these T-cell populations.

Induced CD45 Proximity Potentiates Natural Killer Cell Receptor Antagonism.

Natural killer (NK) cells are a major subset of innate immune cells that are essential for host defense against pathogens and cancer. Two main classes of inhibitory NK receptors (NKR), KIR and CD94/NKG2A, play a key role in suppressing NK activity upon engagement with tumor cells or virus-infected cells, limiting their antitumor and antiviral activities. Here, we find that single-chain NKR antagonists linked to a VHH that binds the cell surface phosphatase CD45 potentiate NK and T activities to a greater extent than NKR blocking antibodies alone in vitro. We also uncovered crosstalk between NKG2A and Ly49 that collectively inhibit NK cell activation, such that CD45–NKG2A and CD45–Ly49 bispecific molecules show synergistic effects in their ability to enhance NK cell activation. The basis of the activity enhancement by CD45 ligation may reflect greater antagonism of inhibitory signaling from engagement of MHC I on target cells, combined with other mechanisms, including avidity effects, tonic signaling, antagonism of weak inhibition from engagement of MHC I on non-target cells, and possible CD45 segregation within the NK cell-target cell synapse. These results uncover a strategy for enhancing the activity of NK and T cells that may improve cancer immunotherapies.

Innate immune responses to three doses of the BNT162b2 mRNA SARS-CoV-2 vaccine.

To explore the effects of SARS-CoV-2-mRNA vaccines on innate immune responses we enrolled 58 individuals who received 3 doses of the BNT162b2 vaccine in a longitudinal study; 45 of these individuals had never been SARS-CoV-2 infected. Results showed that vaccination significantly increased: 1) classical and intermediate inflammatory monocytes, 2) CD56bright, CD56dim, and CD56dim/CD16dim NK cells, and 3) IFN-γ+ ;production as well as perforin and granzyme content by NK cells. Vaccination also reduced expression of the NK inhibitory receptor ILT-2, increasing that of the stimulatory molecule 2DS2. These effects were long-lasting and were boosted by every vaccine dose. Notably, ILT-2 expressing NK cells were reduced even more robustly in COVID-19-recovereed vaccines. BNT162b1 mRNA vaccine is known to induce potent adaptive immune responses; results herein show its ability to modulate innate immune responses as well, offering further support to the indication to proceed with worldwide vaccination efforts to end the SARS-CoV-2 pandemic.

Inhibition of SHP-1 activity by PKC-θ regulates NK cell activation threshold and cytotoxicity.

Natural killer (NK) cells play a crucial role in immunity, killing virally infected and cancerous cells. The balance of signals initiated upon engagement of activating and inhibitory NK receptors with cognate ligands determines killing or tolerance. Nevertheless, the molecular mechanisms regulating rapid NK cell discrimination between healthy and malignant cells in a heterogeneous tissue environment are incompletely understood. The SHP-1 tyrosine phosphatase is the central negative NK cell regulator that dephosphorylates key activating signaling proteins. Though the mechanism by which SHP-1 mediates NK cell inhibition has been partially elucidated, the pathways by which SHP-1 is itself regulated remain unclear. Here, we show that phosphorylation of SHP-1 in NK cells on the S591 residue by PKC-θ promotes the inhibited SHP-1 'folded' state. Silencing PKC-θ maintains SHP-1 in the active conformation, reduces NK cell activation and cytotoxicity, and promotes tumor progression in vivo. This study reveals a molecular pathway that sustains the NK cell activation threshold through suppression of SHP-1 activity.

Control of Memory Phenotype T Lymphocyte Homeostasis: Role of Costimulation.

Foxp3+ T regulatory cells (Tregs), CD4+Foxp3- T cells, and CD8+ T cells are composed of naive phenotype (NP) and memory phenotype (MP) subsets. Ten to 20% of each MP T cell population are cycling (Ki-67+) in vivo. We investigated the contribution of costimulatory (CD28) and coinhibitory (CTLA-4, PD-1) receptors on MP T cell homeostatic proliferation in vivo in the mouse. Blockade of CD28-CD80/CD86 signaling completely abolished MP Tregs and profoundly inhibited MP CD4+Foxp3- T cell proliferation, but it did not affect MP CD8+ T cell proliferation. Marked enhancement of homeostatic proliferation of MP Tregs and MP CD4+Foxp3- T cells was seen after blocking CTLA4-CD80/CD86 interactions and PD-1-PD-L1/2 interactions, and greater enhancement was seen with blockade of both pathways. The CD28 pathway also played an important role in the expansion of Tregs and MP T cells after treatment of mice with agonistic Abs to members of the TNF receptor superfamily, which can act directly (anti-GITR, anti-OX40, anti-4-1BB) or indirectly (anti-CD40) on T cells. Induction of a cytokine storm by blocking the interaction of NK inhibitory receptors with MHC class I had no effect on Treg homeostasis, enhanced MP CD4+ proliferation, and expansion in a CD28-dependent manner, but it enhanced MP CD8+ T cell proliferation in a CD28-independent manner. Because MP T cells exert potent biologic effects primarily before the induction of adaptive immune responses, these findings have important implications for the use of biologic agents designed to suppress autoimmune disease or enhance T effector function in cancer that may have negative effects on MP T cells.

Decidual NK cells kill Zika virus–infected trophoblasts

Zika virus (ZIKV) during pregnancy infects fetal trophoblasts and causes placental damage and birth defects including microcephaly. Little is known about the anti-ZIKV cellular immune response at the maternal-fetal interface. Decidual natural killer cells (dNK), which directly contact fetal trophoblasts, are the dominant maternal immune cells in the first-trimester placenta, when ZIKV infection is most hazardous. Although dNK express all the cytolytic molecules needed to kill, they usually do not kill infected fetal cells but promote placentation. Here, we show that dNK degranulate and kill ZIKV-infected placental trophoblasts. ZIKV infection of trophoblasts causes endoplasmic reticulum (ER) stress, which makes them dNK targets by down-regulating HLA-C/G, natural killer (NK) inhibitory receptor ligands that help maintain tolerance of the semiallogeneic fetus. ER stress also activates the NK activating receptor NKp46. ZIKV infection of Ifnar1 -/- pregnant mice results in high viral titers and severe intrauterine growth restriction, which are exacerbated by depletion of NK or CD8 T cells, indicating that killer lymphocytes, on balance, protect the fetus from ZIKV by eliminating infected cells and reducing the spread of infection.