Expression Of NKp46 Research Articles

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.

Clinicopathological Manifestations and Immune Phenotypes in Adult-Onset Immunodeficiency with Anti-interferon-γ Autoantibodies.

Anti-interferon (IFN)-γ autoantibodies (anti-IFN-γ Abs) is an emerging adult-onset immunodeficiency syndrome. Immune dysfunction in this distinct disorder remains to be clarified. We prospectively collected blood samples of 20 patients with anti-IFN-γ Abs and 40 healthy normal subjects. The percentages of lymphocyte subpopulations, most relevant to T, B, and NK cells, and the percentages of stimulated lymphocytes with cytokine production were assessed using eight-color flow cytometry. The results were adjusted to age and absolute lymphocyte counts. Most (85%) patients presented nontuberculous mycobacterial infection. Skin lesions were predominantly manifested by neutrophilic dermatoses. The involved lymph nodes had granulomatous inflammation, except 22.2% showing atypical lymphoid hyperplasia without granuloma formation. The percentages of CD4 + T cells and nonactivated subpopulations (recent thymic emigrants and naïve subtypes) decreased significantly with increased expression of activation markers and polarization to differentiated cells. The percentage of NK cells increased, but that of two major NK subpopulations, CD161 + CD56bright and CD161 + CD56 + CD16 + subsets, decreased. Increased CD161dim, CD161 + CD56 - CD16 + , and CD57 + NK cell subsets coupled with the decreased expression of NKp30 and NKp46 indicate reconfiguration of the NK cell population and acquisition of adaptive features. Intracellular cytokine production of the lymphocyte subpopulations was significantly low in the patients compared with the control group. We conclude that the immune system in patients with anti-IFN-γ Abs could be exhausted in T cells and be adaptive in NK cells, contributing to the distinct clinicopathologic features.

Evaluation of NKp46 expression and cytokine production of decidual NK cells in women with recurrent pregnancy loss.

PurposeNKp46, a receptor on NK cells, is involved in cytotoxicity and cytokine production. The authors aimed to evaluate the effect of NKp46 on decidual NK (dNK) cells during pregnancy and whether it can be a marker for immunological abnormalities in women with recurrent pregnancy loss (RPL).MethodsFlow‐cytometric analysis was made to assess NKp46 expression and intracellular cytokine production of dNK cells. The proportion of NKp46+ dNK cells was analyzed among RPL patients who aborted karyotypically normal pregnancies and those who either aborted karyotypically abnormal pregnancies or without genetic studies, and controls who were going through the induced abortion.ResultsThe %NKp46+ and %NKp46bright dNK cells were significantly lower in the RPL women who aborted karyotypically normal pregnancies than in the control group. The %NKp46bright dNK cells were significantly correlated with the NK1/NK2 ratio of dNK cells. The %NKp46+ dNK cell cutoff for RPL with immunological abnormalities was determined by the ROC curve analysis. In women with the low %NKp46+ dNK, NK1/NK2 ratios were significantly higher than those with the high.ConclusionRPL patients with an immunological abnormality have decreased NKp46 expression and NK1 shift in dNK cells. NKp46 expression could be a marker for RPL of immunological abnormalities.

Increased NKp44 Expression on CD56dimCD16+ NK Cells in Tumor Microenvironment by Tetrahydropalmatine Derived from Corydalis Yanhusuo W. T. Wang

Increased NKp44 Expression on CD56dimCD16+ NK Cells in Tumor Microenvironment by Tetrahydropalmatine Derived from Corydalis Yanhusuo W. T. Wang

Differential expression of maturation and activation markers on NK cells in patients with active and latent tuberculosis.

NK cells were recently suggested to be important for the initial control of M. tuberculosis infection. The phenotypes of the 3 main NK blood subsets, CD56bright , CD56dim , and CD56neg cells, were characterized by flow cytometry in a cohort of 81 prospectively enrolled subjects (21 untreated patients with active tuberculosis -aTB-, 35 latently TB infected -LTBI- subjects, and 25 non-infected controls), using 9 different mAbs added to whole blood. Compared to LTBI subjects, patients with aTB had lower proportions of total NK cells, lower proportions and numbers of CD56neg cells expressing early maturation markers (CD161, NKp30, NKp46), but higher density of NKp30 and NKp46 expression on both CD56neg and CD56dim subsets, associated with higher expression of granzymes A/B. They also had higher proportions of activated CD69pos cells within all 3 NK cell subsets and, the percentage of CD69pos CD56dim cells among CD69pos and/or NKG2Cpos NK cells was identified as a potential biomarker to discriminate aTB from LTBI. LTBI subjects were in contrast characterized by higher expression of late maturation markers (CD57, KIR molecules) on the CD56neg subset, by higher proportions of NKG2Cpos KIRpos CD56dim NK cells, and by higher in vitro IFN-γ production than patients with aTB. Thus, the in-depth phenotypic characterization of blood NK cell subsets provides new insights on possible functional modifications and the potential role of NK cells in the control of M. tuberculosis infection in humans.

Abstract P191: The combination of CD16A/EGFR bispecific innate cell engager AFM24 with SNK01 NK cells promotes efficacious targeting and killing of EGFR+ tumor cells

Abstract The epidermal growth factor receptor (EGFR) is an overexpressed antigen on several solid cancers resulting in uncontrolled cell proliferation. Current EGFR-targeting agents such as tyrosine kinase inhibitors (TKIs) and monoclonal antibodies (mAbs) mainly rely on EGFR signaling inhibition which is challenged by limited efficacy due to dose limiting toxicity, and/or intrinsic or acquired resistance of the tumor by e.g., KRAS or BRAF mutations. To overcome these limitations and to address the high medical need across EGFR-positive cancers, we investigate a unique approach of combining AFM24, a tetravalent bispecific EGFR- and CD16A-binding innate cell engager (ICE), with the patient-specific NK cell product SNK01. AFM24 mediates high affinity targeting and ADCC of EGFR+ tumor cells by NK cells, independent of EGFR signaling inhibition and irrespective of the mutational status of the tumor. AFM24 is currently being tested as monotherapy in a Phase 1/2a study in patients with EGFR-positive tumors. SNK01 is a first-in-kind, autologous non genetically modified NK cell product with significant anti-tumor cytotoxicity and high and homogeneous expression of CD16A, NKG2D, NKp46 and DNAM-1 , that can be consistently produced even from heavily pre-treated cancer patients. SNK01 showed activity against numerous solid tumors in preclinical studies and in the phase I trials. In order to investigate the synergistic potential of this unique combination, SNK01 cells were generated from PBMCs of healthy individuals and their functional activation by AFM24 in response to EGFR-positive A-431 cells was assessed in cytotoxicity assays and flow cytometry-based CD107a (marker for NK cell degranulation) and IFN-γ mobilization assays. AFM24 binding to and CD16A expression on SNK01 cells was measured by flow cytometry. Both, binding of AFM24 and expression of CD16A were detected on nearly 100% of SNK01 cells, indicating saturation of CD16A-positive SNK01 cells with AFM24. In the presence of AFM24, lysis of EGFR-positive A-431 cells by SNK01 cells was significantly enhanced. Likewise, in response to AFM24, upregulation of CD107a and IFN-γ expression by SNK01 cells was significantly increased upon cross-linking to A-431 cells. By contrast, AFM24 alone did not lead to significant NK cell activation in the absence of target cells or vice versa. Hence, activation of SNK01 cells was strictly dependent on AFM24-mediated cross-linking of SNK01 cells with target cells. In conclusion, this combination holds promise to direct the AFM24-augmented anti-tumor activity of SNK01 cells to EGFR-positive tumor cells independent of EGFR signaling pathway mutations thereby providing a potential new treatment option for underserved patient populations. An IND for a Phase I/II combination trial of AFM24 and SNK01 has been cleared and first-patient-in is on track for the second half of 2021. Citation Format: Jens Pahl, Ting-Ying (Hazel) Cheng, Uwe Reusch, Arndt Schottelius, Wolfgang Fischer, Yong Man Kim, Stephen Chen, Joachim Koch. The combination of CD16A/EGFR bispecific innate cell engager AFM24 with SNK01 NK cells promotes efficacious targeting and killing of EGFR+ tumor cells [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2021 Oct 7-10. Philadelphia (PA): AACR; Mol Cancer Ther 2021;20(12 Suppl):Abstract nr P191.

Low-frequency, exhausted immune status of CD56dim NK cells and disordered inflammatory cytokine secretion of CD56bright NK cells associated with progression of severe HFMD, especially in EV71-infected patients

BackgroundThe roles of CD56bright and CD56dim natural killer (NK) subsets in the viral clearance and inflammatory processes of hand, foot, and mouth disease (HFMD) remain undefined. MethodsA total of 39 HCs and 55 patients were enrolled to analyze peripheral CD56bright and CD56dim NK cells according to cell number, surface receptors, cytotoxic activities, and cytokine production. The plasma concentrations of IL-2, IL-6, IL-10, IFN-γ, TNF-α,and MCP-1 were detected using ELSA. ResultsPeripheral blood NK cells was significantly lower in severe patients than in HCs due to the dramatic loss of CD56dim NK cells with no changes in the cell count of CD56bright NK cells. For mild patients, decreased NKp46 expression coincided with enhanced cytolysis (CD107a, GNLY, and GrB) in CD56dim NK cells and decreased NKG2A expression with enhanced IL-10 production in CD56bright NK cells. In contrast, severe patients showed the dominant expression of NKG2A and decreased expression of NKG2D accompanied by cytotoxic dysfunction in CD56dim NK cells. Imbalanced receptor expression coincided with the increased concentrations of TNF-α in CD56bright NK cells. Moreover, EV71+ patients showed significantly decreased counts of CD56dim NK cells with cytolysis dysfunction, displayed cytokine hypersecretion in CD56bright NK cells, while the EV71− patients displayed significantly higher plasma cytokine concentrations. The changes in the immune function of NK subsets and their subpopulations were closely related to clinical inflammatory parameters. ConclusionsLow-frequency, exhausted immune status of CD56dim NK cells and disordered inflammatory cytokine secretion of CD56bright NK cells were associated with the progression of severe HFMD, especially in EV71-infected patients. This promoted the severity of inflammatory disorders, leading to enhanced disease pathogenesis.

Study of Bone Marrow Mesenchymal Stem Cells Regulating NK Cells Function through Tigit/ CD226 in Patients with Multiple Myeloma

Introduction：Bone marrow microenvironment plays a significant supporting role in the proliferation, differentiation, migration, survival and drug resistance of myeloma cells. To detect the expression of the co-inhibitory receptor TIGIT and the co-stimulatory receptor CD226 on the surface of natural killer cells (NK cells) in multiple myeloma (MM) patients, and changes in the immune phenotypes and killing function of NK cells. Furthermore, to explore the underlying mechanism of bone marrow mesenchymal stem cells (BMSCs) regulating NK cells function through TIGIT/CD226 in patients with MM. The expressions of TIGIT, CD226, activated molecules NKG2D and CD107a and functional molecules IFN-γ and Perforin on the NK cells in bone marrow or peripheral blood were detedted by FCM. BMSCs and NK cells were co-cultured at a ratio of 1:4 in vitro, which was grouped according to different monoclonal antibodies (mAbs) added. After co-cultured, the changes in the expression of NKG2D, NKp30, NKp44 and CD69 of NK cells and IFN-γ concentration were examined by FCM. NK cells and U266 were co-cultured with different E:T ratio and the apoptosis of U266 was detected to evaluate the killing function of NK cells by FCM. The expressions of CD155 on BMSCs in the bone marrow were detected by FCM. The correlations between CD155 expression on BMSCs and the production of IFN-γ and Perforin in bone marrow NK cells, as well as clinical characteristics were analyzed.Results: 1. In bone marrow, expression of TIGIT was significantly higher in patients with NDMM than those in patients with CR and HC (both P values <0.01). CD226 was significantly lower in NDMM patients than those in patients with CR and HC (both P values <0.01). NK cell surface activated molecules NKG2D and CD107a, and functional molecules IFN-γ and Perforin were significantly decreased in NDMM (Fig. A). In peripheral blood, TIGIT, CD226 and above immune phenotype expression levels were basically consistent with the trend in bone marrow. 2. In co-culture experiments of BMSCs and NK cells, only added anti-TIGIT mAbs, compared to both added anti-TIGIT and CD226 mAbs, the immune phenotypes of NK cells significantly increased (Fig. B). And the concentration of IFN-γ in the co-culture supernatants also increased (Fig. C). 3. After co-cultured with BMSCs, NK cells were co-cultured again with U266 in vitro, the apoptosis level of U266 in TIGIT mAbs group increased than those in TIGIT+ CD226 mAbs group at different E: T ratio (Fig. D). 4. The expression of CD155, the ligand of TIGIT and CD226, increased notably on the surface of BMSCs in MM patients(P<0.01) (Fig. E). 5. The expression of CD155 on BMSCs was negatively correlated with the production of IFN-γ and perforin in bone marrow NK cells with NDMM patients. And it has correlation with clinical characteristics including β2-MG, LDH, ALB, Hb, ISS stage and R-ISS stage in patients with MM.Summary: BMSCs may regulate NK cells through TIGIT/CD226. High expression of TIGIT on NK cells may mediate the inhibitory effect of BMSCs in MM patients. Blocking TIGIT could restore NK cells activation and killing function. CD155, a common ligand of TIGIT and CD226, was overexpressed on BMSCs in NDMM patients, but was low expressed on bone marrow MM cells. And it has correlation with NK cells function and clinical characteristics, indicating that CD155 may be involved in the regulation of NK cells function by BMSCs. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Analysis of NKp46 expression and cytokine production in decidual NK cells – Possibility to predict recurrent pregnancy loss

Analysis of NKp46 expression and cytokine production in decidual NK cells – Possibility to predict recurrent pregnancy loss

P10s-PADRE vaccine combined with neoadjuvant chemotherapy in ER-positive breast cancer patients induces humoral and cellular immune responses.

Breast cancer patients diagnosed with HR+/HER2– tumors face a persistent risk of distant recurrence long after completion of their treatment. Strategies to induce anti-tumor immune responses could complement standard-of-care therapies for these patients. The current study was performed to examine the feasibility, safety and immunogenicity of adding P10s-PADRE to standard-of-care chemotherapy in HR+/HER2− early-stage breast cancer patients. Twenty-five subjects were treated in a single-arm Phase Ib clinical trial. Five different immunization schedules were considered to evaluate the feasibility of eliciting an immune response. The primary immunogenicity endpoint was antibody titer. The expression of several activation markers on natural killer (NK) cells and serum concentrations of Th1/Th2 cytokines were also examined. The percentage of tumor-infiltrating lymphocytes (TILs) was determined. Antibody response was superior in schedule C where 3 weekly immunizations preceded the first dose of chemotherapy. A significant change in CD16, NKp46 and CD94 expression levels on NK cells and a rise in serum content of IFN-γ was observed after treatment. Schedule C showed an increase in TILs in residual lesions. The combination therapy is safe and immunogenic with treatment schedule C being immunologically promising. Randomized trials focused on long-term survival outcomes are needed to evaluate clinical benefits.

Sulphation and Hydrolysis Improvements of Bioactivities, and Immuno-Modulatory Properties of Edible Amanita hemibapha Subspecies javanica (Corner and Bas) Mucilage Polysaccharide as a Potential in Personalized Functional Foods.

In this study, the mucilage polysaccharide (MP) from Amanita hemibapha subspecies javanica was prepared by hot water extraction and ethanol precipitation and then fractionated using anion-exchange chromatography equipped with a DEAE Sepharose fast flow column. The most immune-enhancing polysaccharide fraction 2 (MPF2) was subjected to a structural modification such as hydrolysis or over-sulphation. The sulphate and molecular weight (Mw) of over-sulphated (OS1-3) and hydrolysed (HS1-3) derivatives of MPF2 differed between 9.85% and 14.2% and 32.8 and 88.1 × 103 g/mol, respectively. Further, the immune-enhancing properties of MPF2 and its derivatives were tested on RAW264.7 and NK cells through various in vitro assays. Interestingly, a low molecular weight of HS1-3 significantly increased the nitric oxide (NO) production (p < 0.05) more than MPF2, indicating that Mw is a major factor in RAW264.7 cell stimulation. In addition, RAW264.7 cells produced various cytokines by up-regulating mRNA expression levels and the activation of nuclear factor kappa-B (NF-κB) and mitogen-activated protein kinase (MAPK) pathways. On the other hand, OS1-3-treated natural killer (NK) cells induced cytotoxicity in HepG2 cells through the expression of IFN-γ, Grandzyme-B, perforin, NKp30, and FasL. These results demonstrated that sulphate derivatives play an important role in NK cell activation. Further, this study also explores how polysaccharide binds to RAW264.7 and NK cells. MPF2 and HS3 may activate RAW264.7 cells via binding to TLR4 receptors, and OS2 could be activated through the CR3 signalling pathways.

Functions of NK and iNKT cells in pediatric and adult CVID, ataxia telangiectasia and agammaglobulinemia patients

Primary immune deficiencies (PID) are known to be more than 400 genetic defects caused by the impairment in development and/or functions of the immune system. Common Variable Immunodeficiency (CVID), Ataxia Telangiectasia (AT) and Agammaglobulinemia (AG) are examples of the most common immunodeficiency syndrome. Natural killer (NK) cells are a component of innate immune system and play a major role in the host-rejection of both tumors and virally infected cells. iNKT cells have a role in autoimmune and infectious diseases and controlling of tumor rejection. In this study, NK and iNKT cells and their functions, and intracellular cytokine amount are aimed to determine in patients that suffer CVID, AT and AG. NKp30, NKp46, NKG2D, perforin and granzyme mRNA expression levels were analyzed using RT-PCR. Receptors, cytokine amount of NK cell subset and iNKT were analyzed by flow cytometry. Decreased CD3+ T and elevated NK cell subset in pediatric AT were found. Expression of NKp44 was decreased in adult AG, but not in pediatric patients. Low NKp44 expression in CD3−CD16+CD56dim NK cell subset was found in pediatric AT patients. High HLA-DR, perforin and granzyme expression were found in CD3−CD16+CD56dim NK cell subset of pediatric CVID and AT patients. Alteration of the number of NK subsets, NK receptor expression and cytokine production were observed in pediatric patients compared to healthy subjects.

NK cells that differ in expression of NKp46 might play different roles in endometrium

NKp46 is a natural cytotoxicity receptor expressed by NK cells and its expression is decreased in reproductive failure patients. NKp46 can be subdivided into NKp46dim and NKp46bright according to different fluorescence staining intensities. We investigated the role of the NKp46 receptor in determining the reproductive outcomes. Uterine endometrium was collected from 34 women with reproductive failure and divided into the pregnant and failed groups based on the results of a pregnancy reaction test during a 1-year follow-up period. NKp46 receptor and other activating or inhibitory receptors expressed on NK cells as well as intracellular cytokine production by NK cells were analyzed by multicolor flow cytometry. In the failed group, the percentage of NKp46dim NK cells (P < 0.05) was significantly higher and percentages of NKp46bright NK cells (P < 0.01) and CD16−/CD56bright NK cells (P < 0.05) were significantly lower than those in the pregnant group. NKp46dim NK cells were significantly and positively correlated with CD16+/NKp46dim NK cells; NKp46bright NK cells were significantly and positively correlated with CD16−/NKp46bright NK cells. CD16+/NKp46dim NK cells were significantly and positively correlated with IFN-γ- and/or TNF-α-producing NK cells; CD16−/NKp46bright NK cells were significantly and positively correlated with TGF-β1-producing NK cells. We suggest that the NKp46 receptor plays different roles in reproduction based on the different fluorescence intensities associated with NK cells, i.e. NKp46dim NK cells are involved in killing cells, whereas NKp46bright NK cells are involved in cytokine production, indicating that NKp46 could be a predictive marker to see a tolerate condition for embryos.

NK-92 cells change their phenotype and function when cocultured with IL-15, IL-18 and trophoblast cells

NK cell development is affected by their cellular microenvironment and cytokines, including IL-15 and IL-18. NK cells can differentiate in secondary lymphoid organs, liver and within the uterus in close contact with trophoblast cells. The aim was to evaluate changes in the NK cell phenotype and function in the presence of IL-15, IL-18 and JEG-3, a trophoblast cell line. When cocultured with JEG-3 cells, IL-15 caused an increase in the number of NKG2D+ NK-92 cells and the intensity of CD127 expression. IL-18 stimulates an increase in the amount of NKp44+ NK-92 cells and in the intensity of NKp44 expression by pNK in the presence of trophoblast cells. NK-92 cell cytotoxic activity against JEG-3 cells increased only in presence of IL-18. Data on changes in the cytotoxic activity of NK-92 cells against JEG-3 cells in the presence of IL-15 and IL-18 indicate the modulation of NK cell function both by the cytokine microenvironment and directly by target cells. IL-15 and IL-18 were present in conditioned media (CM) from 1st and 3rd trimester placentas. In the presence of 1st trimester CM and JEG-3 cells, NK-92 cells showed an increase in the intensity of NKG2D expression. In the presence of 3rd trimester CM and JEG-3 cells, a decrease in the expression of NKG2D by NK-92 cells was observed. Thus, culturing of NK-92 cells with JEG-3 trophoblast cells stimulated a pronounced change in the NK cell phenotype, bringing it closer to the decidual NK cell-like phenotype.

Serum HBV pregenomic RNA exhibited opposite associations with NKdim and NKbright cell immunity in treatment-naïve chronic hepatitis B patients.

Hepatitis B virus (HBV) pregenomic RNA (pgRNA) is a new biomarker that reflects HBV replication, but its relationship with natural killer (NK) cell immunity in chronic hepatitis B (CHB) is unknown. We assessed serum HBV pgRNA levels in 323 CHB patients by reverse transcription-polymerase chain reaction, assessed cytokine production and activation and inhibitory markers of NK cells by flow cytometry, and measured serum cytokines by enzyme-linked immunosorbent assays (ELISAs). Among the different CHB phases, the serum HBV pgRNA level was highest in the immune-tolerant (IT) and immune-active (IA) phases. Regarding NK and NKdim cells, HBV pgRNA was negatively associated with frequencies, but positively associated with NKp44 and NKp46 expression (activation markers). Regarding NKbright cells, serum HBV pgRNA was positively associated with frequency and programmed cell death protein 1 (PD1) expression (inhibitory marker), but negatively associated with NKp44 and NKp46. Serum HBV pgRNA was not associated with NKp30 (activation marker) on NK cells or subsets. Lastly, serum HBV pgRNA was positively correlated with the levels of serum IL-7 and IL-12P40 (NK cell-promoting cytokines) and negatively correlated with serum prostaglandin E2 (PGE2) level (which negatively regulates NK cells). In conclusion, we found varied relationships between serum HBV pgRNA and NK cells and subsets, indicating that HBV pgRNA may play a complicated role in NK cell-related immunity, providing new information on HBV and host immunity.

Abstract 1609: Peripheral NK-cell immunophenotypes predict progression in nasopharyngeal carcinoma patients

Abstract Objective: The tumorigenesis is closely associated with the alteration in the expression of cell-surface proteins. As NK cells play a vital role in cancer immunotherapy, we thus conducted this study to characterize the immunophenotypes of circulating NK cells and to explore the association between NK phenotypes and disease prognosis. Methods: 249 nasopharyngeal carcinoma (NPC) patients and 11 healthy individuals were recruited in the study, whose peripheral blood samples were collected before treatment. Multi-color flow cytometry analysis was performed. The expression of four inhibitory receptors, PD-1, TIGIT, LAG-3, TIM-3, and four stimulatory receptors, NKp46, NKp30, CD226, NKG2D, were measured in three NK subsets, identified by the co-expression of CD56 and CD16 when gated on CD3-. The LASSO (least absolute shrinkage and selection operator) logistic regression model was used to screen out potential prognostic NK subtypes and calculate the NK-function index. Results: NPC patients exhibited distinct peripheral NK-cell distribution and altered expression of key receptors, as characterized by increased expression of PD-1 and TIGIT on CD56+CD16- cells and decreased expression of NKp46 on CD56+CD16- and CD56-CD16- cells. Four markers were selected from the LASSO model to calculate the NK-function index: PD1 and NKp46 on CD56-CD16+ NK cells, NKp46 on CD56+CD16- cells and NKG2D on CD56+CD16+ cells. Patients with low NK index had significantly shorter progression-free survival (PFS) and distant metastasis-free survival (DMFS), and the NK-function index was an independent prognostic factor for NPC patients. Conclusions: The immunophenotype of peripheral NK cells and its association with survival outcomes of NPC patients revealed its potential as a strong prognosticator for PFS and DMFS, and highlighted the important role of circulating immune environment, especially for the application of immunotherapy. Citation Format: Xiao-Yun Li, Zi-Jian Lu, Hai-Qiang Mai. Peripheral NK-cell immunophenotypes predict progression in nasopharyngeal carcinoma patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1609.

NKp46 Is a Useful Diagnostic Biomarker in Gastrointestinal T-Cell Lymphoproliferative Diseases and Constitutes a Therapeutic Target. a Celac Network Study

Introduction:Primary gastrointestinal (GI) T-cell lymphoproliferative diseases (T-LPD) are heterogeneous entities, which diagnoses and therapeutic options remain to be standardized. Beside aggressive lymphoma, such as enteropathy-associated T-cell lymphoma (EATL) and monomorphic epitheliotropic intestinal T-cell lymphoma (MEITL), the indolent T-cell lymphoproliferative disease (indolent T-LPD) has been recently described. A differential diagnosis to consider is type II refractory coeliac disease (RCDII) that may complicate CD. Intraepithelial lymphocytes (IEL) from RCDII are dependent for survival on IL-15, which reprograms T-lymphocytes towards a cytotoxic NK phenotype. We previously showed that NKp46 is a new biomarker for both diagnosis and prognosis in GI T-LPD (ICML-2017 Lugano, unpublished).Patients and Methods:The expression of NK receptors was assessed on CD/RCD by flow-cytometry. The number of NKp46 positive IEL per 100 epithelial cells (NKp46+ IEL/100 EC) was studied by immunohistochemistry (IHC) on 195 biopsies from 84 CD/RCD, 44 GI T-cell lymphoma, and on a blinded confirmatory cohort of 48 CD/RCD.8B6A-PBD is an anti-NKp46 monoclonal antibody conjugated (ADC) to a potent cytotoxic drug, pyrrolobenzodiazepine (PBD). For in vivo assessment, tumor-bearing NOD-SCID mice were randomly assigned to PBS, isotype-matched irrelevant Ig-PBD controls, or 8B6A-PBD treatment groups. For ex vivo cytotoxicity assays, isolated cells from fresh small bowel biopsies of RCDII patients were incubated with 8B6A-PBD, Ig-PBD, anti-NKp46 deglycosylated or PBD alone.Results:NKp46 was the only NK receptor that differs between RCDII and CD or RCDI. By doing ROC analysis, a number of NKp46+ IEL/100 EC above 25 clearly discriminated RCDII from CD and RCDI, with excellent positive and negative predictive values of 100 and 95% respectively. Among CD and RCD patients, the survival was shorter if this count is above 25 (OS-5years 72.8% vs. 96.4%, P=0.0004). NKp46 was expressed in EATL (n=20/25) and MEITL (n=4/4) but was never expressed in indolent T-LPD (n=0/15). Positive NKp46 GI T-cell lymphoma had a shorter survival compared to negative NKp46 cases (OS-5years 5.4% vs. 50.5%, P=0.0011).In vivo the anti-NKp46-PBD promoted tumor regression (Fig. B) and prolonged survival (Fig. A) of NOD-SCID mice injected by human NKp46-transfected Raji tumors embedded in matrigel. Ex vivo and as a proof of concept, the anti-NKp46-PBD was highly potent (Fig. D) and selective (Fig. C) against NKp46 positive primary sorted IEL (n=5).Conclusion:NKp46 provides a new biomarker for both diagnosis and prognosis in GI T-LPD. We propose a diagnostic algorithm for GI T-LPD based on the NKp46 expression, which is adapted to immediate, individual patient management. Overall, our results demonstrate for the first time that anti-NKp46 ADC may serve as an effective therapy for RCDII, EATL and MEITL and provide strong preclinical rationale for evaluation of 8B6A-PBD in clinical studies. [Display omitted] DisclosuresCheminant:Innate Pharma: Research Funding. Bruneau:Innate Pharma: Research Funding. Malamut:Cellimmune: Research Funding. Pouyet:MI-mAbs: Employment. Lhospice:Innate Pharma: Employment. Molina:Merck Serono: Honoraria; Takeda: Other: travel support to the ASH meeting; Novartis: Honoraria. Cellier:Cellimmune: Research Funding. Cerf-Bensussan:Cellimmune: Research Funding. Hermine:Novartis: Research Funding; Celgene: Research Funding; AB Science: Equity Ownership, Honoraria, Patents & Royalties, Research Funding; Hybrigenics: Research Funding; INatherys: Equity Ownership, Research Funding.

Combination of Lenalidomide and Rituximab (R2) Targets Immune Dysfunction in FL By Activating T and NK Cells Ex Vivo and Is Differentiated from Chemotherapy

▪Introduction: Follicular lymphoma (FL) is an indolent disease characterized by a dysfunctional immune compartment. The standard of care in FL is chemotherapy-based treatment (R-CHOP/R-CVP or R-Bendamustine), with a high overall response rate of >90% but low complete response (CR) rates between 25% to 31% (Flinn 2014). The majority of patients still relapse and often experience immune suppression resulting from chemo-related cytotoxicity that may ultimately impact outcomes. Lenalidomide (Len) has both potent direct antineoplastic and immunomodulatory activity. Len in combination with rituximab (Rtx) has shown enhanced antibody-dependent cellular cytotoxicity (ADCC) and direct apoptosis in B-cell lymphoma in vitro and in animal models (Wu 2008; Hernandez-Ilizaliturri 2005). Multiple phase II studies showed that R2 therapy achieves clinical synergy in treatment naive (Fowler 2014; Martin ICML 2017) and relapsed/refractory settings (Leonard 2015; Andorsky ASCO 2017). Here we provide a mechanistic rationale beyond ADCC for the activity observed in the clinical setting using patient samples. We show T and NK cell activation by Len in FL patients ex vivo and demonstrate R2activity in FL cell lines providing clear differentiation from immunochemotherapy.Methods: Peripheral blood mononuclear cells (PBMCs) from FL patients were treated with Len (1nM-10µM), analyzed for cytokine production by ELISA, T and NK-cell activation on day 3 and proliferation of T and NK-cells on day 5 by flow. Len, idelalisib, bendamustine (1nM-10µM), and ibrutinib (0.1nM-1µM) were each used in combination with Rtx in an ADCC and cell autonomous assay to measure apoptosis by Annexin V/ToPro-3 flow cytometry against FL cell lines DoHH2 and RL. For cell autonomous assay, cells were treated with therapeutic agents for 7 days followed by 24hr Rtx treatment. Synergy of Rtx in combination with each therapeutic agent was calculated using fractional product method (Chou, 1984). For ADCC assays, healthy donor (HD) or FL PBMC were treated with therapeutic agent for 3 days followed by a 4-hr incubation with Rtx-treated CFSE labeled target tumor cells. PBMC viability was measured immediately prior to co-culture by Annexin V/To-Pro.Results: In ex vivo FL PBMCs, Len significantly enhanced proliferating CD4+and CD8+ T cells by up to 3 and 7-fold respectively (p<0.001) and proliferating NK and NKT by up to 2 and 5-fold vs DMSO. In FL PBMCs, Len significantly increased the production of TNF-α, IFN-γ and GMCSF up to 4, 6 and 10-fold respectively (p≤0.01) vs DMSO. Len increased IL-2 and RANTES by up to 3-fold vs DMSO. Len activates NK cells by a 2-fold increase in expression of CD56, CD25, NKp30, OX40 and Granzyme B in FL PBMCs vs DMSO. Len significantly enhanced expression of CD154, HLA-DR, CD278 and CD134 on CD8+T cells by up to 2-fold (p≤0.05) vs DMSO. The R2 combination was more potent than R-chemo in Rtx mediated ADCC. Combination of Len (1µM) and Rtx (1µg/mL) significantly increased ADCC to 44% (p≤0.01) in DOHH2 and RL cells vs Rtx alone (4% in DoHH2 and 17% in RL) using HD PBMCs. Combination of Len (1µM) and Rtx (10ng/mL) significantly increased ADCC by 41% in DOHH2 and 31% in RL (p≤0.05) vs Rtx alone (12% in DoHH2 and 21% in RL cells) using FL PBMCs. Combination of Rtx with idelalisib, ibrutinib and bendamustine demonstrated no effect or an antagonistic effect on Rtx mediated ADCC against FL cells. In direct apoptosis assay, combination of Len (1µM) and Rtx (10µg/mL) was significantly synergistic and increased apoptosis to 77% (p≤0.0001) vs Len (29%) or Rtx alone (40%). In RL cells, R2 was significantly additive and increased apoptosis by 55% (p≤0.001) vs Len (37%) or Rtx alone (34%). No synergy was observed with Rtx in combination with the other chemo agents in ADCC assays. In PBMC viability assays, Len had no effect whilst idelalisib (2µM), ibrutinib (1µM) and bendamustine (10µM) significantly decreased viability by 25, 37 and 46 % respectively (p≤0.01-0.05).Conclusion: R2 is a novel and effective combination immunotherapy in FL. The mechanism of action of this combination results in T and NK cell activation, which leads to enhanced ADCC and addresses the known immune dysfunction in FL. This combination is clearly differentiated from chemo-immunotherapy. The R2 combination immunotherapy is currently being assessed in the FL frontline phase 3 RELEVANCE trial (NCT01476787) and in the relapsed FL setting in the AUGMENT trial (NCT01938001). DisclosuresChiu:Celgene: Employment, Equity Ownership. Hagner:Celgene: Employment, Equity Ownership. Trisal:Celgene: Employment, Equity Ownership. Waldman:Celgene: Employment, Equity Ownership, Patents & Royalties: US 9,587,281 B2. Cavanaugh:Celgene: Employment. Gandhi:Celgene Corporation: Employment, Equity Ownership.

Effect of Immunomodulatory Drugs (IMiDs) on Immune Effectors after Autologous Stem Cell Transplantation (ASCT) in Multiple Myeloma

INTRODUCTION: Patients with multiple myeloma have several phenotypic and functional immune aberrations that suppress host anti-myeloma immune responses. The degree and type of immune dysfunction can be targeted and reversed in many patients with active treatment. Therapies that result in deep and sustained remissions play a part in immunological control of disease in long-term responders. IMiD-mediated immunomodulatory effects conferring immune system reactivation have been reported in MM in the maintenance setting. We hypothesized that the circulating effector immune cell profile post-ASCT will be impacted by depth of response and IMiD based maintenance therapy.METHODS: Thirty multiple myeloma patients who underwent ASCT and IMiD based maintenance and consented for specimen collection protocol were included in this study. Blood and bone marrow samples were collected before starting IMiD based maintenance therapy on Day 60+ post ASCT (time point defined as baseline). Blood samples were then serially collected after 1 month (n=24), 3 months (n=26) and 6 months (n=19) of maintenance treatment. Multicolor flow cytometry was used for NK, NK-T and T cell distribution/activation immunotyping, and for measurement of bone marrow minimal residual disease (MRD) status. Threshold for MRD positivity was established as 1.5x10-5 abnormal/clonal plasma cells. Clinical response assessment was performed post-ASCT at baseline per IMWG guidelines. Welch's t-test was used to compare each immune variable between MRDpos and MRDneg groups and one-way ANOVA was used to analyze changes of each immune variable over time. The association of individual immune variable with progression free survival (PFS) was assessed by Cox regression analysis.RESULTS: In total, 16 patients achieved CR/sCR, 13 VGPR and 1 PR. MRD status was determined for 20 of 30 patients, 5 were MRDneg and 15 MRDpos. MRDpos patients had an immunotype characterized by higher expression of KIR2DS4 on NK (p=0.002) and NK-T cells (p=0.048) at baseline, compared with MRDneg patients. Twenty-six patients received single agent IMiD maintenance, 4 received IMiD/proteasome inhibitor combination. During IMiD maintenance therapy, NK and NK-T cell acquired phenotypes associated with greater effector functions as shown by increase in NK NKG2D+ (p=0.04), NK Tim3+ (p=0.049) and NK-T Tim3+ (p=0.01). T cells were marked by a NKG2D loss (p<0.001) and Tim3 gain (p<0.001) of expression. The median follow-up of the cohort was 19.3 months (IQR 14.8-21.3). Seven patients (5 MRDpos, 2 MRD status unknown) relapsed within 12 to 24 months post-ASCT. High KIR2DS4 (HR=1.072, 95% CI: 1.019-1.128; p=0.008), NKp46 (HR=1.05, 95% CI: 1.004-1.098; p=0.031) and NKG2A (HR=1.222, 95% CI: 1.018-1.466; p=0.032) expression by NK-T cells at baseline were associated with shorter PFS. NK and NK-T cells retained higher KIR2DS4 expression after 1, 3 and 6 months of IMiD maintenance therapy in relapsing patients compared with those in remission (Figure1).CONCLUSIONS: Our results demonstrate that KIR2DS4 expression by NK and NK-T cells is associated with MRD status post-transplant, as well as PFS in multiple myeloma patients treated with IMiD maintenance therapy. This simple blood-based immunotype, if validated in larger studies, could be used for longitudinal assessment to predict early on after starting IMiD maintenance treatment whether to continue this treatment or change to different treatment. Patients predicted to derive less clinical benefit from IMiDs could be offered alternative treatment regimens such as monoclonal antibodies, other immunotherapies or epigenetic therapy. [Display omitted] DisclosuresBhutani:Amgen: Speakers Bureau; Prothena Therapeutics: Research Funding; BMS: Speakers Bureau; Takeda Oncology: Speakers Bureau. Voorhees:Amgen: Speakers Bureau; Janssen: Consultancy, Speakers Bureau; Celgene: Consultancy, Speakers Bureau; Novartis: Consultancy; Takeda: Consultancy; Bristol-Myers Squibb: Consultancy; Oncopeptides: Consultancy. Usmani:Array BioPharma: Honoraria, Research Funding; Onyx: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Bristol-Myers Squibb: Honoraria, Research Funding; Pharmacyclics: Honoraria, Research Funding; Takeda: Consultancy, Honoraria, Research Funding, Speakers Bureau; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Sanofi: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Amgen: Consultancy, Honoraria, Speakers Bureau; Novartis: Speakers Bureau; Skyline: Honoraria, Membership on an entity's Board of Directors or advisory committees; Millennium: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau.

Live cell imaging of highly activated natural killer cells against human hepatocellular carcinoma in vivo

Background aimsTracking administered natural killer (NK) cells in vivo is critical for developing an effective NK cell-based immunotherapy against human hepatocellular carcinoma (HCC). Here the authors established a new molecular imaging using ex vivo-activated NK cells and investigated real-time biodistribution of administered NK cells during HCC progression. MethodsEx vivo-expanded NK cells from healthy donors were labeled with a near-infrared lipophilic cytoplasmic dye, and their proliferation, surface receptor expression and cytotoxicity activity were evaluated. Human HCC HepG2 cells were implanted into the livers of NOD.Cg-Prkdcscid IL2rgtm1Wjl/SzJ (NSG) mice. The authors administered 1,1’-dioctadecyltetramethyl indotricarbocyanine iodide (DiR)-labeled NK cells intravenously to non-tumor-bearing and intrahepatic HCC tumor-bearing NSG mice. Fluorescent imaging was performed using a fluorescence-labeled organism bioimaging instrument. Single cell suspensions from the resected organs were analyzed using flow cytometry. ResultsThe fluorescent DiR dye was nontoxic and did not affect the proliferation or surface receptor expression levels of the NK cells, even at high doses. The administered DiR-labeled NK cells immediately migrated to the lungs of the non-tumor-bearing NSG mice, with increased NK cell signals evident in the liver and spleen after 4 h. NK cells migrated to the intrahepatic tumor-bearing livers of both early- and late-stage HCC mice within 1 h of injection. In early-stage intrahepatic tumor-bearing mice, the fluorescence signal increased in the liver until 48 h post-injection and decreased 7 days after NK injection. In late-stage HCC, the NK cell fluorescence signal was the highest in the liver for 7 days after NK injection and persisted for 14 days. The purity of long-term persistent CD45+CD56+CD3− NK cells was highest in early- and late-stage HepG2-bearing liver compared with normal liver 2 weeks after NK injection, whereas highest purity was still observed in the lungs of non-tumor-bearing mice. In addition, Ki-67 expression was detected in migrated human NK cells in the liver and lung up to 72 h after administration. With HepG2 tumor progression, NK cells reduced the expression of NKp30 and NKG2D. ConclusionsAdministered NK cells were successfully tracked in vivo by labeling the NK cells with near-infrared DiR dye. Highly expanded, activated NK cells migrated rapidly to the tumor-bearing liver, where they persisted for 14 days after administration, with high purity of CD45+CD56+CD3− NK cells. Liver biodistribution and persistence of administered NK cells showed significantly different accumulation patterns during HCC progression.