NKG2D Ligands Research Articles

Valproic Acid Increased CAR T Cell Cytotoxicity Against Acute Myeloid Leukemia

Background The treatment paradigm of B cell malignancies has dramatically changed in recent years with the introduction of immunotherapy, especially CAR-T cell therapy. However, only limited efficacy was observed in AML with CAR-T cell therapy. Methods In this study, the molecular, clinical, and tumor immune microenvironments characteristics of AML patients in TCGA were performed on an online bio-information analysis website. The expressions of CD123 and CLL-1 on the AML cells from clinical samples were analyzed by flow cytometry. Then we constructed anti-CD123 CAR and CLL-1 CAR with different co-stimulation domains CD28 or 4-1BB and detected the anti-AML effects. To increase the efficacy of CAR-T cell therapy, we tested different combination strategies, such as checkpoint inhibitor PD-1/PD-L1 antibodies and histone deacetylase inhibitor (HDACi) in vivo and in vitro. RNA sequencing and western blot were performed to analyze the potential mechanism of valproic acid (VPA) in upregulating NKG2DL. Results The data suggested that CD123 and CLL-1 were highly expressed on AML leukemia cells (p=0.0031, p=0.0069). The proportion of various immune cells such as T cell subsets and NK cells in AML patients was significantly lower than that in healthy donors. The process of immune infiltration mainly stagnated in the recruitment and activation of immune cells or the tracking migration of immune cells. The immunosuppressive molecule PD-1 was negatively correlated with memory T cells, while the active molecules CD27, CD28, and NKG2D were positively correlated with CD8 + memory T cells. Both CD123 CAR and CLL-1 CAR displayed specific anti-AML effects. To improve the lysis effects of CAR-T cells, we combined CAR-T cell therapy with different agents. PD-1/PD-L1 antibody could only slightly improve the potency of CAR-T cell therapy (CD123 CAR-T 60.92±2.9087% VS 65.43±2.1893%, 60.92±2.9087% VS 67.43± 3.4973%, CLL-1 CAR-T 37.37±3.908% VS 41.89 ±5.1568%, 37.37±3.908% VS 42.84± 4.2635%). While, one of HDACi, valproic acid (VPA) could synergize with CAR-T cell therapy and significantly improve CAR-T cell potency against AML cells, especially in pre-treated with VPA for 48h ( CLL-1 CAR-T 34.97±0.3051% VS 88.167±1.5327% p<0.0001, CD123 CAR-T 26.87±2.7010% VS 82.56±3.086%, p<0.0001 in MV411, CLL-1 CAR-T 78.77±1.2061% VS 93.743±1.2333% p<0.0001, CD123 CAR-T 64.10±1.5130% VS 94.427±0.142%, p=0.0001 in THP-1). What's more, the anti-AML effects of combination therapy were also observed in the AML mice model. NCG mice were injected with 1×106 THP-1 cells that express firefly luciferase and GFP label (THP-1-Fluc) via tail vein, 1 week later mice were infused with VPA 0.5mM (iv, n=6), VPA 100mg/kg (ip, n=6), VPA 0.5mM (iv, twice a day, n=6) and PBS (iv, n=6), then CD123 CAR-T cells or untreated T cells were infused 48h later. The combination therapy prolonged the overall survival of mice when compared to single CAR-T cell therapy (median survival:180 days VS unfollowed). One of the possible mechanisms underlying we found was that NKG2D could upregulate on activated CD8+T cells, and VPA could upregulate NKG2D ligand expression in the AML cells which may contribute to NKG2D-mediated cytotoxicity of CAR-T cells against tumor cells. In addition, the expression of HDAC2 and H4 in THP-1 cells was significantly decreased after VPA treatment, suggesting that VPA may enhance the expression of NKG2DL by degrading HDAC2 expression. Conclusion CD123 and CLL-1 are promising targets for AML CAR-T cell therapy. A combination of VPA pre-treatment and CAR-T against AML exhibits synergic effects, which may provide a promising strategy in the clinical treatment of R/R AML. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

A Novel Dual-Antigen Targeting Approach Enables Off-the-Shelf CAR NK Cells to Effectively Recognize and Eliminate the Heterogenous Population Associated with AML

While acute myeloid leukemia (AML) treatment has improved, relapse is still common, even after reduced intensity conditioning allogeneic transplantation. Natural killer (NK) cells are a promising allogeneic cell immunotherapy without the risk of graft-versus-host disease or cytokine release syndrome. We and others have reported that haploidentical NK cells can safely result in 30-50% complete remissions in advanced AML patients when given with high-dose lymphodepleting chemotherapy and exogenous cytokine support. However, while NK cell natural cytotoxicity has a clinical signal in AML, increased specificity and multi-dosing are likely to enhance the efficacy and durability of this treatment strategy. A number of stress ligands, including the MHC-I polypeptide-related sequence A and B (MICA/B), bind to the NK cell activating receptor NKG2D and initiate NK cell killing of transformed and infected cells, and several groups have shown promising results in clinical trials by developing NK cells that carry a NKG2D chimeric antigen receptor (CAR). However, the distal α1 and α2 domains of MICA/B that are recognized by NKG2D can be proteolytically cleaved from the surface of the tumor by metalloproteases, allowing for escape from NK cell-mediated detection and elimination. Wucherpfennig and colleagues have described that the membrane proximal α3 domain remains on the cell surface and may be a potential target for immunotherapy across many cancers, including AML. However, since AML is heterogenous, we hypothesized that clinical success would require dual-targeting and selected CD33 as the ideal complementary targeting approach since CD33 is a validated marker expressed on greater than 80% of AML blasts. To this end, our lab has developed a Tri-specific Killer Engager (TriKE) capable of agonistically ligating the CD16 Fc receptor to CD33 found on AML along with IL-15 co-stimulation to further activate the NK cell response. Here, we demonstrate the efficacy of a multiplexed-gene edited induced pluripotent stem-cell (iPSC) derived NK cell (iNK) product that expresses a high-affinity, non-cleavable version of CD16 (hnCD16), a membrane-bound IL-15 fusion receptor, CD38 knockout to enhance metabolic fitness, and a CAR against the α3 domain of MICA/B (α3 MICA/B) to drive a potent response against AML alone and when combined with anti-CD33 TriKE. In the initial study, the iNK cell backbone (iNK cells without the α3 MICA/B CAR) induced potent activity against the AML cell line HL60, and displayed further enhancement of activity with the addition of anti-CD33 TriKE (GTB-3650), representing combined effects of natural cytotoxicity and antibody-dependent cellular cytotoxicity. To show specificity for α3 MICA/B targeting, the AML cell line THP-1 was stained for the presence of MICA/B by flow cytometry using the 6D4 clone that recognizes the α1/2 domains of MICA/B or the 7C6 clone that uniquely recognizes the α3 domain +/- proteolytic cleavage with trypsin (Fig. 1A-B). Significant expression of the α3 domain was observed on THP-1 cells using the 7C6 antibody, which was highly expressed even after protease treatment. On the other hand, the α1/2 domains of MICA/B were detectable at lower levels using the 6D4 antibody and were undetectable after protease treatment (Fig. 1A-B). To assess a dual targeting approach, THP-1 cells were used as targets in live imaging functional assays under standard conditions (Effector:Target [E:T] 2:1). The iNK cell backbone had modest natural cytotoxicity at 3 hours that was significantly enhanced by the addition of α3 MICA/B CAR. The effect was further improved with more rapid killing kinetics when combined with the anti-CD33 TriKE (Fig. 1C). To evaluate the efficacy of dual-targeting in a more stringent physiologic manner, we mimicked "stress” conditions by minimizing the E:T ratio to 0.25:1. While all effector conditions induced immediate killing in 4 hours at the low E:T ratio, sustained tumor control was only observed with dual-antigen targeting (Fig. 1D). Studies with primary AML targets, +/- preincubation of decitabine and all-trans retinoic acid, known to upregulate NKG2D ligands in AML, are in progress and will be discussed. In summary, dual-targeting strategies using off-the-shelf CAR NK cells targeting α3 MICA/B in combination with antigen-specific TriKE targeting CD33 represent an ideal clinical strategy to enhance efficacy and durability of treatment in advanced AML. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

IMMU-16. SOMATIC MUTATIONS IN HUMAN LEUKOCYTE ANTIGEN GENES AND ANTIGEN PRESENTATION PATHWAY GENES IN MALIGNANT GLIOMA

Abstract Immunotherapeutic approaches in cancer aim to booster T cell mediated immune responses. Tumor-specific T cells recognize target cells via human leukocyte antigen (HLA) molecules before cell specific lysis is initiated. Malignant gliomas are known for their immunoevasive and immunosuppressive properties which impede effective tumor-specific immune responses, including clinical responses to immune checkpoint inhibition. Our aim was to investigate whether somatic mutation in HLA genes and antigen presentation pathway genes are present in malignant gliomas. METHODS We performed next generation sequencing in 138 patients, including matched tumor and blood samples from 100 IDHwt glioblastomas, 25 IDHmut astrocytomas and 13 IDHmut oligodendrogliomas. Sequencing was performed on tumor and peripheral blood for HLA class I (HLA-A, -B, -C), HLA class II (HLA-DR, -DP, -DQ), non-classical HLA molecules (HLA-E, -F, -G, -H), MICA/B, TAP1/2 and beta2 microglobulin (B2M). Identified calls were validated using targeted nanopore sequencing. RESULTS In total, we identified 28 mutations based on Illumina sequencing. Mutations were located in HLA-B (2 mutations), HLA-C (1), B2M (2), HLA-DQA1 (4), HLA-DQB1 (4), HLA-DPB1 (1), HLA-E (1), HLA-F (2), HLA-G (3), HLA-H (1), MICA (3), MICB (2), TAP1 (1) and TAP2 (1). No mutations were found in HLA-A and HLA-DRB1. A frequency cutoff of 5% was applied. Additional mutations (n = 46) below this cutoff were identified. The mutations were found in 30 patients (21.7%). Some patients harbored up to 7-8 mutations in multiple HLA and antigen presenting pathway genes. Most mutations were found in the MICA (MHC class I polypeptide–related sequence A) gene locus, which is engaged by the NKG2D ligand which is broadly expressed on NK cells, gd and ab T cells. CONCLUSION Our study demonstrates that somatic mutations in HLA and antigen presentation pathway genes are frequently present in malignant gliomas.

TMIC-72. ALL-TRANS RETINOIC ACID INDUCES DURABLE TUMOR IMMUNITY IN IDH MUTANT GLIOMAS AND CIRCUMVENTS REPRESSION OF CRBP1-RETINOIC ACID AXIS

Abstract Diffuse gliomas are fatal, immunologically cold tumors characterized by mutations in isocitrate dehydrogenase 1 or 2 (IDH1/2) genes. IDH mutations have been shown to result in global genomic hypermethylation and drastically altered transcriptomic landscape. We and others have shown that IDH mutation is direct responsible for the immunological cold landscape observed in IDH mutated glioma. Using a combination of animal models and patient-derived glioma stem cells (GSCs), we identify retinoic acid signaling as a primary regulator of innate and adaptive immune activation in IDHm gliomas. Using the Sleeping Beauty (SB) orthotopic murine model of IDH glioma, which faithfully recapitulates the TME seen in patients, we show that intracellular accumulation of oncometabolite 2-HG leads to hypermethylation and transcriptional repression of Retinol Binding Protein-1 (RBP1), which encode CRBP1, a key chaperone protein required for intracellular all-trans-retinoic-acid biosynthesis in glial cells. Restoration retinoic acid signaling through the addition of all-trans retinoic acid (ATRA) resulted in spontaneous tumor regression and prolonged survival. ATRA administration is further correlated with increased CD8, NK, M1 macrophages and decreased Treg and MDSC populations within the TME. Single-cell sequencing of the TME shows increased type I interferon signaling, myeloid cell polarization, T cell activation, and TCR diversity. Using knockdown and overexpression techniques in patient-derived GSCs, we show that CRBP1 activity is critical for NK mediated immune surveillance and cytotoxicity through regulation of NKG2D ligand expression and immune cell migration through differential regulation of CXCL12 and CCL2. In summary, epigenetic silencing of RBP1 by 2-HG induced hypermethylation leads to a global immune dysregulation resulting in an immunologically cold landscape. Restoration of retinoic acid signaling is a promising immunotherapeutic approach for the treatment of IDHm gliomas.

Tetra-arsenic tetra-sulfide enhances NK-92MI mediated cellular immunotherapy in all-trans retinoic acid-resistant acute promyelocytic leukemia.

Acute promyelocytic leukemia (APL) is liable to induce disseminated intravascular coagulation and has a high early mortality. Although the combination of all-trans retinoic acid (ATRA) and arsenic trioxide (ATO) has significantly improved the complete remission rate, there are still some patients developed drug resistance. Growing evidence suggests that natural killer (NK) cell-mediated immunotherapy as a new treatment can help slow the progression of hematological malignancies. Previous studies also indicated that some tumors exhibited excellent responsiveness to NK cells in vitro. However, many clinical trial results showed that the anti-tumor effect of NK cells infusion alone was not ideal, which may be related to the inactivation of infiltrating NK cells caused by strong immunosuppression in tumor microenvironment, but the specific mechanism remains to be further explored. In the present study, we demonstrated that low doses of tetra-arsenic tetra-sulfide (As4S4) not only enhanced the in vitro killing of NK-92MI against ATRA-resistant APL cells, but also strengthened the growth inhibition of xenografted tumors in APL mouse model. Mechanistically, As4S4 altered the expression of natural killer group 2 member D ligands (NKG2DLs) and cytokines in APL cells, and PD-1 in NK-92MI cells. In addition, database retrieval results further revealed the relationship between the differentially regulated molecules of As4S4 and immune infiltration and its impact on prognosis. In conclusion, our findings confirmed the potential of As4S4 as an adjuvant for NK-92MI in the treatment of ATRA-resistant APL.

A signature based on NKG2D ligands to predict the recurrence of hepatocellular carcinoma after radical resection.

Due to the high recurrence, the HCC prognosis remains poor. Yet, the biomarkers for predicting the recurrence of high-risk patients are currently lacking. We aimed to develop a signature to predict the recurrence of HCC based on NKG2D ligands. The multivariate Cox proportional hazards regression was used to select recurrence-related variables of NKG2D ligands in HCC patients from The Cancer Genome Atlas (TCGA). HCC patients from the OEP000321 dataset and Guilin cohort were used to validate the predictive signature. The mRNA expression of NKG2D ligands was measured by QRT-PCR. Immunohistochemistry analysis of HCC tissue microarray samples was used to identify the expression of NKG2D ligands. In this study, NKG2D ligands expression in the mRNA and protein level was both abnormally expressed in HCC and associated with recurrence-free survival (RFS). Then, the recurrence-related variables of NKG2D ligands in HCC were selected by the multivariate Cox proportional hazards regression. Among the eight NKG2D ligands, MICA (HR=1.347; 95% CI=1.012-1.793; p=0.041), ULBP3 (HR=0.453; 95% CI=0.231-0.889; p=0.021) and ULBP5 (HR=3.617; 95% CI=1.819-7.194; p < 0.001) were significantly correlated with RFS in the TCGA-LIHC cohort. Then, the signature was constructed by the three NKG2D ligands. The predictive effectiveness of this signature was also validated in the OEP000321 dataset and Guilin cohort. Further, HCC patients were classified into low-risk and high-risk subgroups by the predictive score. Compared with the low-risk group, the high-risk group had poor RFS in both training and validation cohorts. Importantly, compared with the low-risk patients with the G1-G2 stage, the levels of infiltrated NK-activated cells and NKG2D expression were both lower in the high-risk patients. The signature based on MICA, ULBP3, and ULBP5 could predict HCC recurrence.

Enhanced natural killer cell anti-tumor activity with nanoparticles mediated ferroptosis and potential therapeutic application in prostate cancer

Ferroptosis provides an opportunity to overcome the cancer cell therapeutic resistance and modulate the immune system. Here an interaction between ferroptosis of cancer cells and natural killer (NK) cells was investigated with a clinical grade iron oxide nanoparticle (ferumoxytol) for potential synergistic anti-cancer effect of ferroptosis and NK cell therapy in prostate cancer. When ferumoxytol mediated ferroptosis of cancer cells was combined with NK cells, the NK cells’ cytotoxic function was increased. Observed ferroptosis mediated NK cell activation was also confirmed with IFN-γ secretion and lytic degranulation. Upregulation of ULBPs, which is one of the ligands for NK cell activating receptor NKG2D, was observed in the co-treatment of ferumoxytol mediated ferroptosis and NK cells. Additionally, HMGB1 and PD-L1 expression of cancer cells were observed in the treatment of ferroptosis + NK cells. Finally, in vivo therapeutic efficacy of ferumoxytol mediated ferroptosis and NK cell therapy was observed with significant tumor volume regression in a prostate cancer mice model. These results suggest that the NK cells’ function can be enhanced with ferumoxytol mediated ferroptosis.

Romidepsin Enhances the Killing Ability of NKG2D-CAR-T Cells through Enhanced Expression of NKG2DL against Ovarian Cancer Cells

Background: Upregulating tumor cell targeting antigens and improving the cytotoxicity of chimeric antigen receptor T cell (CAR-T) are expected to facilitate better treatment efficacy for solid cancers represented by ovarian cancer. Methods: Killer cell lectin-like receptor subfamily K member 1 ligands (NKG2DL) are the target antigens for ovarian cancer. NKG2D-CAR-T cells were constructed for NKG2D ligand on the ovarian cancer cell surface. We used flow cytometry to evaluate the expression of NKG2DL on SKOV3 (a human ovarian cancer adenocarcinoma cell line). Innovatively, when combined with romidepsin to treat ovarian cancer cell SKOV3, to evaluate the killing ability of the combined strategy, we verified the cytotoxicity of CAR-T cells by lactate dehydrogenase (LDH) release test and determined the secretion of cytokines by enzyme-linked immuno sorbent assay (ELISA). Results: The results of flow cytometry showed effective activation of the NKG2D-CAR-T cells, and romidepsin treatment resulted in increased expression of NKG2DL on the surface of SKOV3. Cytotoxicity test showed that romidepsin could enhance the killing ability of NKG2D-CAR-T cells against ovarian cancer cells by regulating their NKG2DL expression (p &lt; 0.05). The killing effects and secretion of interferon-γ (IFN-γ) increased synchronously (p &lt; 0.05). Levels of interleukin-2 (IL-2) and Pore-forming protein (PFP) were statistically significant at a low target ratio but programmed cell death protein 1 (PD-1) remained unaffected (p ≥ 0.05). Conclusions: Enhancing the expression of tumor target antigen is a solution to improve the limited application of CAR-T cells in solid cancers.

Efficacy and Safety of Inosine Pranobex in COVID-19 Patients: A Multicenter Phase 3 Randomized Double-Blind, Placebo-Controlled Trial.

Inosine pranobex (IP), an immunomodulatory agent, is used in the treatment of various viral infections. The results of a phase 3 randomized controlled trial are reported, evaluating the efficacy and safety of IP in the treatment of mild to moderate COVID‐19. It includes 416 symptomatic patients with confirmed SARS‐CoV‐2 infection. In addition to a defined standard of care, patients randomly (1:1) receive either IP 500 mg tablet (IP group) or a matching placebo (placebo group) at 50 mg kg−1 body weight/day rounded to the nearest 500 mg dose (maximum 4 g day−1) administered in 3–4 divided doses for 10 days. Compared to the placebo group, IP group shows significantly higher rates of clinical response (CR) and clinical cure (CC) on Day‐6 for both non‐hospitalized patients and the total population. IP group shows significantly earlier CR and CC with fewer adverse events and no mortality. Based on these findings and the fact that IP increases natural killer cell‐mediated cytotoxicity of virus‐infected cells as an early immune response to viral infection and enhances NKG2D ligand expression, it is concluded that IP should be started early to maximize the benefit in mild to moderate COVID‐19 patients. (Trial registration number: CTRI/2021/02/030892).

Radiosensitization effect by HDAC inhibition improves NKG2D-dependent natural killer cytotoxicity in hepatocellular carcinoma.

BackgroundHepatocellular carcinoma (HCC) is one of the leading causes of cancer-related death worldwide. Radiotherapy (RT) controls HCC unsatisfactorily and temporarily. Histone deacetylase inhibitor (HDACi) is a heterogeneous group of epigenetic therapeutics with promising anticancer effects and synergism in combination with RT. HDACi modulates natural killer (NK) cell ligand expression on tumor cells, and leads to immune evasion of cancer cells. Expressions of NK group 2D (NKG2D) ligands on cancer cells determine the cytotoxic effect by interacting with NKG2D receptor on NK cells. However, the role of NKG2D signaling in HCC upon combined RT and HDACi remains unclear.Method In vitro co-culture system with NK cells was tested for human and murine HCC cell lines. Pan-HDACi (panobinostat) and specific HDAC4 knockdown (HDAC4-KD) were used for HDAC inhibition. Clonogenic assay and flow cytometry examined HCC cell survival and NKG2D ligand expression, respectively. Syngeneic mouse model was used to validate the radiosensitizing effect in vivo.ResultsCombined RT and HDACi/HDAC4-KD significantly enhanced NK cell-related cytotoxicity and increased NKG2D ligands, MICA/MICB expressions in human and RAE-1/H60 expressions in murine HCC cells. Delayed tumor growth in vivo by the combinational treatment of RT and HDACi/HDAC4-KD was shown with the associated NKG2D ligand expressions. However, NKG2D receptor did not significantly change among tumors.ConclusionRadiosensitizing effect with combined RT and HDAC inhibition increased the expression of NKG2D ligands in HCC cells and enhanced their susceptibility to NK cell-mediated cytotoxicity. These findings imply the potential use of combined RT/HDACi and NK cell-directed immunotherapy.

MMP-2 Inhibitor-Mediated Tumor Microenvironment Regulation Using a Sequentially Released Bio-Nanosystem for Enhanced Cancer Photo-Immunotherapy.

Combining photodynamic therapy (PDT) with natural killer (NK) cell-based immunotherapy has shown great potential against cancers, but the shedding of NK group 2, member D ligands (NKG2DLs) on tumor cells inhibited NK cell activation in the tumor microenvironment. Herein, we assembled microenvironment-/light-responsive bio-nanosystems (MLRNs) consisting of SB-3CT-containing β-cyclodextrins (β-CDs) and photosensitizer-loaded liposomes, in which SB-3CT was considered to remodel the tumor microenvironment. β-CDs and liposomes were linked by metalloproteinase 2 (MMP-2) responsive peptides, enabling sequential release of SB-3CT and chlorin e6 triggered by the MMP-2-abundant tumor microenvironment and 660 nm laser irradiation, respectively. Released SB-3CT blocked tumor immune escape by antagonizing MMP-2 and promoting the NKG2D/NKG2DL pathway, while liposomes were taken up by tumor cells for PDT. MLRN-mediated photo-immunotherapy significantly induced melanoma cell cytotoxicity (83.31%), inhibited tumor growth (relative tumor proliferation rate: 1.13% of that of normal saline) in the xenografted tumor model, and enhanced tumor-infiltrating NK cell (148 times) and NKG2DL expression (9.55 and 16.52 times for MICA and ULBP-1, respectively), achieving a synergistic effect. This study not only provided a simple insight into the development of new nanomedicine for programed release of antitumor drugs and better integration of PDT and immunotherapy but also a novel modality for clinical NK cell-mediated immunotherapy against melanoma.

414.6: Porcine ULBP1 Disruption Does Not Significantly Reduce Human NK Cell Activation and Cytotoxicity in an in Vitro Model

Background: Pig-to-human xenotransplantation has the potential to address the critical organ shortage. Genetically-engineered pig organs have significantly prolonged survival in human and nonhuman primates by overcoming hyperacute rejection. Human natural killer (NK) cell-mediated acute xenograft rejection of porcine endothelial cells has been established. Porcine UL-Binding Protein (ULBP1) has been identified as a porcine ligand of human NK cell activating receptor NKG2D; interactions presumably lead to NK cell activation and ultimately porcine cell cytotoxicity. We sought to improve pig-to-human compatibility by eliminating ULBP1 ligand in an immortalized porcine liver-derived endothelial cell line (ipLDEC) with five-gene knockout (5GKO). The 5GKO cell line has mutations in GGTA1/CMAH/β4galNT2/SLA-I α-chain/β2M: three genes encoding enzymes responsible for xenoantigen production and two genes encoding swine leukocyte antigen class I. Methods: CRISPR/Cas9 technology was used to knockout the porcine ULBP1 gene in the 5GKO cell line to create a ULBP1-KO/5GKO cell line. Human peripheral blood mononuclear cells (PBMCs) from four donors were activated with human IL-2 for five days. Human PBMCs were co-cultured with 5GKO, ULBP1-KO/5GKO, and Wild-Type (WT) ipLDECs. Cells were stained with antibodies to CD45, CD3, CD56, and CD107a. Flow cytometry analysis was gated on the CD3-CD56+ NK cell population and monitored for expression of CD107a, a marker of NK cell degranulation. Calcein AM cytotoxicity assay was performed to determine human NK cell-mediated ipLDECs cell death. WT, 5GKO, ULBP1-KO/5GKO, HLA-G+/5GKO (positive control) were incubated in a calcein solution for 30 minutes at a concentration of 106 cells/mL. Stained porcine cells were co-cultured with activated human PBMCs at a 10:1 E:T ratio for 4 hours. Co-culture supernatant was harvested and calcein release was measured at an excitation wavelength of 485 nm and emission wavelength of 530 nm to calculate cytotoxicity percentages. Results: Porcine ULBP1 gene mutation was confirmed by Sanger sequencing (Figure 1a), establishing a porcine ULBP1-KO/5GKO cell line. There is no statistically significant difference in NK cell activation between co-cultures of human PBMCs (n=4) with pULBP1-KO/5GKO and 5GKO ipLDEC using CD107a. (Figure 1b). There is no statistically significant difference in NK-induced cytotoxicity between co-cultures of human PBMCs (n=6) with porcine ULBP1-KO/5GKO and 5GKO cell lines using Calcein AM assays (Figure 1c). As expected, HLA-G+/5GKO cytotoxicity was decreased (p = 0.0168) compared to WT (Figure 1c). Conclusion: We established a porcine ULBP1-KO/5GKO cell line. Our studies demonstrate no statistically significant difference in NK cell activation and NK cell-mediated cytotoxicity between 5GKO and ULBP1-KO/5GKO cell lines. Our studies show that ULBP1 is not a crucial ligand for human NKG2D. Future studies will target different human NK cell activating porcine ligands.

Isolation of a Natural Killer Group 2D Small-Molecule Ligand from DNA-Encoded Chemical Libraries.

Natural Killer Group 2D (NKG2D) is a homo-dimeric transmembrane protein which is typically expressed on the surface of natural killer (NK) cells, natural killer T (NKT) cells, gamma delta T (γδT) cells, activated CD8 positive T-cells and activated macrophages. Bispecific molecules, capable of bridging NKG2D with a target protein expressed on the surface of tumor cells, may be used to redirect the cytotoxic activity of NK-cells towards antigen-positive malignant T-cells. In this work, we report the discovery of a novel NKG2D small molecule binder [KD =(410±60) nM], isolated from a DNA-Encoded Chemical Library (DEL). The discovery of small organic NKG2D ligands may facilitate the generation of fully synthetic bispecific adaptors, which may serve as an alternative to bispecific antibody products and which may benefit from better tumor targeting properties.

NKp44-based chimeric antigen receptor effectively redirects primary T cells against synovial sarcoma

BackgroundT-cell receptor-engineered T-cell therapies have achieved promising response rates against synovial sarcoma in clinical trials, but their applicability is limited owing to the HLA matching requirement. Chimeric antigen receptor (CAR) can redirect primary T cells to tumor-associated antigens without requiring HLA matching. However, various obstacles, including the paucity of targetable antigens, must be addressed for synovial sarcoma. Ligands for natural killer (NK) cell-activating receptors are highly expressed by tumor cells. MethodsThe surface expression of ligands for NK cell-activating receptors in synovial sarcoma cell lines was analyzed. We analyzed RNA sequencing data deposited in a public database to evaluate NKp44-ligand expression. Primary T cells retrovirally transduced with CAR targeting NKp44 ligands were evaluated for their functions in synovial sarcoma cells. Alterations induced by various stimuli, including a histone deacetylase inhibitor, a hypomethylating agent, inflammatory cytokines, and ionizing radiation, in the expression levels of NKp44 ligands were investigated. Results: Ligands for NKp44 and NKp30 were expressed in all cell lines. NKG2D ligands were barely expressed in a single cell line. None of the cell lines expressed NKp46 ligand. Primary synovial sarcoma cells expressed the mRNA of the truncated isoform of MLL5, a known cellular ligand for NKp44. NKp44-based CAR T cells specifically recognize synovial sarcoma cells, secrete interferon-γ, and exert suppressive effects on tumor cell growth. No stimulus altered the expression of NKp44 ligands. ConclusionNKp44-based CAR T cells can redirect primary human T cells to synovial sarcoma cells. CAR-based cell therapies may be an option for treating synovial sarcomas.

Circular EZH2-encoded EZH2-92aa mediates immune evasion in glioblastoma via inhibition of surface NKG2D ligands

Glioblastoma (GBM) is a highly aggressive primary brain tumour and is resistant to nearly all available treatments, including natural killer (NK) cell immunotherapy. However, the factors mediating NK cell evasion in GBM remain largely unclear. Here, we report that EZH2-92aa, a protein encoded by circular EZH2, is overexpressed in GBM and induces the immune evasion of GBM stem cells (GSCs) from NK cells. Positively regulated by DEAD-box helicase 3 (DDX3), EZH2-92aa directly binds the major histocompatibility complex class I polypeptide-related sequence A/B (MICA/B) promoters and represses their transcription; it also indirectly represses UL16-binding protein (ULBP) transcription by stabilizing EZH2. The downregulation of NK group 2D ligands (NKG2DLs, including MICA/B and ULBPs) in GSCs mediates NK cell resistance. Moreover, stable EZH2-92aa knockdown enhances NK cell-mediated GSC eradication in vitro and in vivo and synergizes with anti-PD1 therapy. Our results highlight the immunosuppressive function of EZH2-92aa in inhibiting the NK cell response in GBM and the clinical potential of targeting EZH2-92aa for NK-cell-directed immune therapy.

CD277 agonist enhances the immunogenicity of relapsed/refractory acute myeloid leukemia towards Vδ2+ T cell cytotoxicity.

Relapse and refractoriness remain the major obstacles in clinical treatment of acute myeloid leukemia (AML). Efficacy of current therapeutic strategies for relapsed/refractory (R/R) AML is generally unsatisfying. Vδ2+ T cells have become an attractive candidate for immunotherapy of various types of tumors. However, the results were not exciting in some pilot studies utilizing Vδ2 cell-based protocols to treat R/R AML. Functional receptors on Vδ2 cells and immunogenic ligands on leukemia cells are both critical to the anti-AML effect of Vδ2 cells, which have not been characterized in the context of R/R AML. CD277 can bind to phosphoantigens and promote the activation of Vδ2 cells. Anti-CD277 (clone 20.1) monoclonal antibody (20.1 mAb) has been identified as an agonist of CD277. Whether 20.1 mAb sensitizes R/R AML cells awaits investigation. Herein, we showed that the expressions of activating receptors on Vδ2 cells and CD277 on leukemia cells were deficient in patients with R/R AML. While agonists for NKG2D and TRAIL ligands did not increase the immunogenicity of R/R AML cells, 20.1 mAb significantly enhanced the cytotoxicity of Vδ2 cells on the drug-resistant human AML cell line and different types of primary AML cells from R/R patients. The sensitizing effect of 20.1 mAb was dependent on inducing degranulation of Vδ2 cells. These findings suggest a decisive role of CD277 in mediating the recognition of R/R AML cells by Vδ2+ T cells. CD277 agonist combining adoptive transfer of Vδ2+ T cells may improve the efficacy in the treatment of R/R AML.

Upregulation of NKG2D ligands impairs hematopoietic stem cell function in Fanconi anemia.

Fanconi anemia (FA) is the most prevalent inherited bone marrow failure (BMF) syndrome. Nevertheless, the pathophysiological mechanisms of BMF in FA have not been fully elucidated. Since FA cells are defective in DNA repair, we hypothesized that FA hematopoietic stem and progenitor cells (HSPCs) might express DNA damage–associated stress molecules such as natural killer group 2 member D ligands (NKG2D-Ls). These ligands could then interact with the activating NKG2D receptor expressed in cytotoxic NK or CD8+ T cells, which may result in progressive HSPC depletion. Our results indeed demonstrated upregulated levels of NKG2D-Ls in cultured FA fibroblasts and T cells, and these levels were further exacerbated by mitomycin C or formaldehyde. Notably, a high proportion of BM CD34+ HSPCs from patients with FA also expressed increased levels of NKG2D-Ls, which correlated inversely with the percentage of CD34+ cells in BM. Remarkably, the reduced clonogenic potential characteristic of FA HSPCs was improved by blocking NKG2D–NKG2D-L interactions. Moreover, the in vivo blockage of these interactions in a BMF FA mouse model ameliorated the anemia in these animals. Our study demonstrates the involvement of NKG2D–NKG2D-L interactions in FA HSPC functionality, suggesting an unexpected role of the immune system in the progressive BMF that is characteristic of FA.

Does immune destruction drive all forms of bone marrow failure?

Current paradigms of bone marrow failure (BMF) pathophysiology suggest that immune-mediated destruction of hematopoietic stem and progenitor cells (HSPCs) drives acquired aplastic anemia. In contrast, loss of HSPCs due to senescence and/or apoptosis causes BMF in inherited BMF syndromes. In this issue of the JCI, Casado and colleagues challenge this dichotomous conception by demonstrating that NK cell–dependent, immune-mediated hematopoietic suppression and HSPC clearance drive BMF in Fanconi anemia (FA). They show that genotoxic stress upregulates natural killer group 2 member D ligands (NKG2D-L) on FA HSPCs leading to NK cell cytotoxicity through NKG2D receptor activation. Inhibition of NKG2D–NKG2D-L interactions enhanced FA HSPC clonogenic potential and improved cytopenias in vivo. These results provide alternative targets for the development of immunosuppressive therapies to reduce HSPC loss and mitigate the risk of hematologic malignancies in FA.

GAS6/TAM signaling pathway controls MICA expression in multiple myeloma cells.

NKG2D ligands play a relevant role in Natural Killer (NK) cell -mediated immune surveillance of multiple myeloma (MM). Different levels of regulation control the expression of these molecules at cell surface. A number of oncogenic proteins and miRNAs act as negative regulators of NKG2D ligand transcription and translation, but the molecular mechanisms sustaining their basal expression in MM cells remain poorly understood. Here, we evaluated the role of the growth arrest specific 6 (GAS6)/TAM signaling pathway in the regulation of NKG2D ligand expression and MM recognition by NK cells. Our data showed that GAS6 as well as MERTK and AXL depletion in MM cells results in MICA downregulation and inhibition of NKG2D-mediated NK cell degranulation. Noteworthy, GAS6 derived from bone marrow stromal cells (BMSCs) also increases MICA expression at both protein and mRNA level in human MM cell lines and in primary malignant plasma cells. NF-kB activation is required for these regulatory mechanisms since deletion of a site responsive for this transcription factor compromises the induction of mica promoter by BMSCs. Accordingly, knockdown of GAS6 reduces the capability of BMSCs to activate NF-kB pathway as well as to enhance MICA expression in MM cells. Taken together, these results shed light on molecular mechanism underlying NKG2D ligand regulation and identify GAS6 protein as a novel autocrine and paracrine regulator of basal expression of MICA in human MM cells.

DNAM-1-chimeric receptor-engineered NK cells, combined with Nutlin-3a, more effectively fight neuroblastoma cells in vitro: a proof-of-concept study.

Adoptive transfer of engineered NK cells, one of clinical approaches to fight cancer, is gaining great interest in the last decade. However, the development of new strategies is needed to improve clinical efficacy and safety of NK cell-based immunotherapy. NK cell-mediated recognition and lysis of tumor cells are strictly dependent on the expression of ligands for NK cell-activating receptors NKG2D and DNAM-1 on tumor cells. Of note, the PVR/CD155 and Nectin-2/CD112 ligands for DNAM-1 are expressed primarily on solid tumor cells and poorly expressed in normal tissue cells. Here, we generated human NK cells expressing either the full length DNAM-1 receptor or three different DNAM-1-based chimeric receptor that provide the expression of DNAM-1 fused to a costimulatory molecule such as 2B4 and CD3ζ chain. Upon transfection into primary human NK cells isolated from healthy donors, we evaluated the surface expression of DNAM-1 and, as a functional readout, we assessed the extent of degranulation, cytotoxicity and the production of IFNγ and TNFα in response to human leukemic K562 cell line. In addition, we explored the effect of Nutlin-3a, a MDM2-targeting drug able of restoring p53 functions and known to have an immunomodulatory effect, on the degranulation of DNAM-1-engineered NK cells in response to human neuroblastoma (NB) LA-N-5 and SMS-KCNR cell lines. By comparing NK cells transfected with four different plasmid vectors and through blocking experiments, DNAM-1-CD3ζ-engineered NK cells showed the strongest response. Furthermore, both LA-N-5 and SMS-KCNR cells pretreated with Nutlin-3a were significantly more susceptible to DNAM-1-engineered NK cells than NK cells transfected with the empty vector. Our results provide a proof-of-concept suggesting that the combined use of DNAM-1-chimeric receptor-engineered NK cells and Nutlin-3a may represent a novel therapeutic approach for the treatment of solid tumors, such as NB, carrying dysfunctional p53.