NK Cell Expression Research Articles

Gene expression analysis of sarcoma-infiltrating NK cells identifies signatures correlating with favorable prognosis and potential new targets for augmenting NK function

Abstract Success of current T-cell based immunotherapies in soft tissue sarcomas (STS) is limited while pre-clinical and clinical studies have shown evidence of NK activity in STS. To investigate the effects of the tumor microenvironment (TME) on tumor-infiltrating NK cell gene expression, we isolated circulating and tumor-infiltrating NK and CD3 T cells from sarcoma patients undergoing surgery (N=4) and performed RNA sequencing analysis. Comparing Differential Gene Expression (DGE) of flow-sorted intra-tumoral NK cells to circulating NK cells, we identified upregulation of genes involved in mitogen signaling inhibition (DUSP4) and metabolic function (SMPD3, SLC7A5), but not of genes typically associated with cytotoxic function (e.g. IFNG, GZMB). In contrast, intra-tumoral T cells showed significant upregulation of both activating (CD137) and inhibitory genes (TIM-3) compared to circulating T cells. We then queried the TCGA STS dataset and stratified cases into “NK Activating TME” and “NK Suppressing TME” based on the CIBERSORT NK signature analysis program (based on high or low proportion of activated NK cells, respectively). Patients with NK Activating TME had significantly improved survival compared to NK Suppressive TME (log-rank p = 0.03). DGE identified unique tumor-associated gene ontology between NK activated versus suppressive subsets with upregulation of the retinoic acid pathway. Unlike T cells which demonstrate significant DGE between circulating and tumor-infiltrating subsets, NK cells appear to have similar gene expression between blood and tumor. Therefore, although NK cells appear to be prognostic in STS, their anti-tumor effects may be dictated by systemic rather than local factors.

Brain tumor T cells inhibited by their natural KLR(B1) instinct.

Charting the gene-expression landscape of glioma-infiltrating T cells demonstrates that these T cells can express NK cell receptors, which inhibit the killing of glioma cells.

A novel Cytochrome P450 26A1 expressing NK cell subset at the mouse maternal-foetal interface.

Cyp26a1 had important roles in mouse embryo implantation and was highly expressed in some of NK cells at the human maternal‐foetal interface in early pregnancy. However, the regulatory effect of Cyp26a1 on NK cells remains poorly understood. Through qPCR and flow cytometric assays, we found that Cyp26a1 was expressed by mouse uterine NK cells but not spleen NK cells during the peri‐implantation period and there was a group of NK cells that highly expressed Cyp26a1, that is Cyp26a1+NK cell subset. single cell‐population transcriptome sequencing on Cyp26a1+NK and Cyp26a1−NK cell subsets was performed. We found that there were 3957 differentially expressed genes in the Cyp26a1+NK cell subset with a cut‐off of fold change ≥2 and FDR < 0.01, 2509 genes were up‐regulated and 1448 genes were down‐regulated in Cyp26a1+NK cell subset. Moreover, cytokine‐cytokine receptor interaction signalling pathway and natural killer cell–mediated cytotoxicity signalling pathway were enriched according to KEGG pathway enrichment analysis. We further found that the expression of Gzma and Klrg1 was significantly increased and Fcgr4 was significantly decreased when inhibiting Cyp26a1. Our experimental results show that there is a novel NK cell subset of Cyp26a1+NK cells in mouse uterus and Cyp26a1 can regulate the gene expression of Gzma, Klrg1 and Fcgr4 in the Cyp26a1+NK cells.

Low-dose interleukin-2-loaded nanoparticle effect on NK and T-reg cell expression in experimentally induced type 1 diabetes mellitus.

IntroductionType 1 diabetes mellitus is an autoimmune disorder characterized by inflammatory damage to pancreatic β cells resulting in loss of insulin secretion. In autoimmune type 1 diabetes mellitus (T1D) natural killer cells (NK) initiate pancreatic islets cell lyses in autoimmune T1D. Loss of T regulatory cells (Treg) at disease onset facilitates the activation and accumulation of NKs in the pancreatic microenvironment. A proper low-dose interleukin 2 (IL-2) could enhance Tregs and enforce control and regulation of pro-inflammatory NKs.AimThis relation needs to be studied to improve therapeutic strategies aimed at resetting the balance between Tregs and proinflammatory cells.Material and methodsWe used novel formulations of low-dose IL-2 loaded on chitosan nanoparticles. The study included 116 T1D BALB/c mice experimentally induced by streptozotocin, divided into groups. Their splenocytes were maintained in a short-term culture for assessment of expression of CD4+FOXP3+ Treg and NKp46+NK by both flow cytometry and enzyme-linked immunoassay (ELISA). Morphological, immunohistochemical, and morphometrical analyses were done. In vitro suppressor assay was used to assess the suppressor effect of Treg cells after exogenous IL-2 treatment.ResultsNK cell expression, NKp46 level, and NK cell functions were modulated more in mice injected with IL-2-loaded chitosan nanoparticles than in other groups. A statistical inverse correlation was found between Treg and NK cell expression in IL-2-loaded chitosan with 0.3 µIU (p = 0.047), and this correlation was related to FOXP3 expression on Treg cells. The modified expression of NK and NKp46 was noticed in mice injected with 0.3 µIU for longer duration (3 weeks) (p < 0.001), but the NK functions did not show any significant changes with prolonged treatment.ConclusionsProlonged administration of low-dose IL-2 results in the vigorous expression of NKp46, indicating a significant role of Tregs in NK stimulation and motivation. Low-dose IL-2 selectively modulates NKp46 NK and FOXP3+ Tregs and increases their expression.

Therapy of lymphoma by immune checkpoint inhibitors: the role of T cells, NK cells and cytokine-induced tumor senescence

BackgroundAlthough antibodies blocking immune checkpoints have already been approved for clinical cancer treatment, the mechanisms involved are not yet completely elucidated. Here we used a λ-MYC transgenic model of endogenously...

Persons who inject drugs (PWID) retain functional NK cells, dendritic cell stimulation, and adaptive immune recall responses despite prolonged opioid use.

Previous literature suggests that acute opioid use results in the functional impairment of the immune response, thereby decreasing resistance to viral infection. Here, we assessed if innate and adaptive immune responses are compromised ex vivo in persons who inject drugs (PWID) and whether long-term injection drug use may impact host susceptibility to in vitro HIV infection. We measured the frequency, activation state, and functional profile of NK cells, dendritic cells, and CD4+ and CD8+ T cells in low-risk PWID who do not share needles, high-risk needle-sharing PWID, and control donors who did not inject drugs. We also assessed plasma levels of inflammatory markers and CD4+ T cell susceptibility to HIV infection. We observed a significant increase in the amount of sCD14 (P=0.0023, n=16) and sCD163 (P=0.0001, n=16) in the plasma of PWID compared to controls. Evidence of constitutive activation was noted in PWID as compared to controls with increased CD69 expression in CD56dim NK cells (P=0.0103, n=26) and increased CD38 and HLA-DR expression in CD4+ T cells (P=0.0355, n=23). However, no innate or adaptive functional differences were detected between PWID and controls, including: NK cell direct or antibody-dependent cellular cytotoxicity poly-functional response, TLR-stimulated dendritic cell/NK crosstalk, CD8+ T cell response to Staphylococcal enterotoxin B or CMV/EBV/FLU peptides, or constitutive or anti-CD3/CD28-stimulated CD4+ T cell infectivity with CCR5-tropic or CXCR4-tropic HIV-1 isolates. Our data indicate that PWID who utilize opioids over as prolonged time frame can retain a functional ex vivo immune response without a measurable increase in CD4+ T cell infectivity suggesting that leukocytes from PWID are not intrinsically more susceptibility to infection with HIV than non-PWID controls.

Impact of Long-Term Cryopreservation on Blood Immune Cell Markers in Myalgic Encephalomyelitis/Chronic Fatigue Syndrome: Implications for Biomarker Discovery.

Myalgic Encephalomyelitis/Chronic Fatigue Syndrome (ME/CFS) is a complex neuroimmune disorder characterized by numerous symptoms of unknown etiology. The ME/CFS immune markers reported so far have failed to generate a clinical consensus, perhaps partly due to the limitations of biospecimen biobanking. To address this issue, we performed a comparative analysis of the impact of long-term biobanking on previously identified immune markers and also explored additional potential immune markers linked to infection in ME/CFS. A correlation analysis of marker cryostability across immune cell subsets based on flow cytometry immunophenotyping of fresh blood and frozen PBMC samples collected from individuals with ME/CFS (n = 18) and matched healthy controls (n = 18) was performed. The functionality of biobanked samples was assessed on the basis of cytokine production assay after stimulation of frozen PBMCs. T cell markers defining Treg subsets and the expression of surface glycoprotein CD56 in T cells and the frequency of the effector CD8 T cells, together with CD57 expression in NK cells, appeared unaltered by biobanking. By contrast, NK cell markers CD25 and CD69 were notably increased, and NKp46 expression markedly reduced, by long-term cryopreservation and thawing. Further exploration of Treg and NK cell subsets failed to identify significant differences between ME/CFS patients and healthy controls in terms of biobanked PBMCs. Our findings show that some of the previously identified immune markers in T and NK cell subsets become unstable after cell biobanking, thus limiting their use in further immunophenotyping studies for ME/CFS. These data are potentially relevant for future multisite intervention studies and cooperative projects for biomarker discovery using ME/CFS biobanked samples. Further studies are needed to develop novel tools for the assessment of biomarker stability in cryopreserved immune cells from people with ME/CFS.

Tgfβ-Imprinting Decrease CD38 Expression and Lead to Metabolic Reprogramming on Primary NK Cell

Transforming growth factor-β (TGF-β) plays an essential role in regulating immune responses through its immunossupressive effect on adaptive and innate immune cells. In addition, we and others have shown that alterations in cell-intrinsic metabolism profiles can significantly impact the function of immune cells. Previously, our group demonstrated that TGFβ-imprinting (TGFβi) decreases NK cell sensitivity to TGFβ through SMAD3 suppression and enhances a pro-inflammatory phenotype with hypersecretion of IFN-γ, TNF-α, and GM-CSF, which appeared to be mediated by an epigenetic mechanism. To evaluate this process, we conducted RNA-seq and ATAC-seq to evaluate the impact of chromosomal remodeling on gene expression. In addition to confirming low SMAD3 transcription, this data showed that TGFβi NK cells have decreased CD38 expression, which we confirmed at the protein level. Since CD38 is an ectoenzyme that regulates nicotinamide adenine dinucleotide (NAD+), a critical component of OXPHOS in both T and NK cells, we examined the effect of TGFβi on NK cell metabolism. TGFβi primary NK cells were generated from peripheral blood of healthy donors as previously described, and using a mitochondrial stress test assay, we observed higher oxygen consumption rates (OCR). However, in the glycolysis stress test assay we observed comparable extracellular acidification rates (ECAR) between Standard expanded and TGFbi NK cells, resulting in higher OCR/ECAR ratios in TGFbi NK cells. Since TGFβ has been shown to induce fragmented mitochondria, and inhibition of mitochondrial fragmentation improved mitochondrial metabolism, we evaluated subcellular structure of TGFβi NK cellsby transmission electron microscopy (TEM). We found no difference in mitochondrial fragmentation between STD and TGFβi NK cells. Together, these results show that TGFβi induces epigenetic reprograming of NK cells that results in increased OXPHOS through CD38 suppression. Moreover, the decreases CD38 expression in TGFβi NK cells could be a promising non-genetic alternative to generating CD38-negative NK cells to avoid fratricide in combination with DARA Figure Disclosures Lee: Kiadis Pharma Netherlands B.V: Consultancy, Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees, Patents &amp; Royalties.

PRDM1 decreases sensitivity of human NK cells to IL2-induced cell expansion by directly repressing CD25 (IL2RA).

IL2 receptor signaling is crucial for human NK cell activation and gain of effector functions. The molecular mechanisms involved in termination of IL2 activation are largely unknown in human NK cells. PR/SET domain 1 was previously reported to decrease cell growth and increase apoptosis in an IL2-dependent manner in malignant NK cell lines, suggesting the possibility of down-regulation of IL2 signaling pathway gene(s) through direct transcriptional repression. Using ChIP-Seq, we identified a PRDM1 binding site on the first intron of CD25 (IL2RA), which codes for the IL2 receptor subunit regulating sensitivity to IL2 signaling, in primary NK cells activated with engineered K562 cells or IL2. Ectopic expression of PRDM1 down-regulated CD25 expression at transcript and protein levels in two PRDM1 nonexpressing NK cell lines. shRNA-mediated knockdown of CD25 in two malignant NK cell lines led to progressive depletion of NK cells in low IL2 concentrations. By contrast, ectopic CD25 expression in primary human NK cells led to progressive increase in cell number in CD25-transduced cells in low IL2 concentrations. Altogether these results reveal a pivotal role of PRDM1 in inhibition of IL2-induced NK cell expansion through direct repression of CD25 in activated human NK cells. These observations provide additional support for the role of PRDM1 in attenuation of NK cell activation and growth, with implications on neoplastic transformation or NK cell function when it is deregulated.

Dynamics of NK, CD8 and Tfh cell mediated the production of cytokines and antiviral antibodies in Chinese patients with moderate COVID-19.

Recent studies have demonstrated a marked decrease in peripheral lymphocyte levels in patients with coronavirus disease 2019 (COVID‐19) caused by severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2). Few studies have focused on the changes of NK, T‐ and B‐cell subsets, inflammatory cytokines and virus‐specific antibodies in patients with moderate COVID‐19. A total of 11 RT‐PCR‐confirmed convalescent patients with COVID‐19 and 11 patients with non‐SARS‐CoV‐2 pneumonia (control patients) were enrolled in this study. NK, CD8+ T, CD4+ T, Tfh‐like and B‐cell subsets were analysed using flow cytometry. Cytokines and SARS‐CoV‐2‐specific antibodies were analysed using an electrochemiluminescence immunoassay. NK cell counts were significantly higher in patients with COVID‐19 than in control patients (P = 0.017). Effector memory CD8+ T‐cell counts significantly increased in patients with COVID‐19 during a convalescent period of 1 week (P = 0.041). TIM‐3+ Tfh‐like cell and CD226+ Tfh‐like cell counts significantly increased (P = 0.027) and decreased (P = 0.022), respectively, during the same period. Moreover, ICOS+ Tfh‐like cell counts tended to decrease (P = 0.074). No abnormal increase in cytokine levels was observed. The high expression of NK cells is important in innate immune response against SARS‐CoV‐2. The increase in effector memory CD8+ T‐cell counts, the up‐regulation of inhibitory molecules and the down‐regulation of active molecules on CD4+ T cells and Tfh‐like cells in patients with COVID‐19 would benefit the maintenance of balanced cellular and humoural immune responses, may prevent the development of severe cases and contribute to the recovery of patients with COVID‐19.

Effects of Regulating Smad7 Gene on Epithelial-Mesenchymal Transition in Keloid Keratinocyte

The effect of Smad7 on epithelial-Mesenchymal Transition (EMT) of keloid keratinocytes was studied. Culture formed keloid cutin cells (KK) and normal skin cutin cell (NK cells), built the Smad7 too slow virus slow virus vector and Smad7 interference expression vector, screening the best expression and interfering with the slow virus infection NK and KK cells respectively, and contrast carrier puro screening stable expression cell lines, stem cells can be divided into 8 groups: NK-Control (normal training of NK cells); NK-NC (NK cells screened against lentivirus); NK-shSmad7 (NK cells that interfere with lentivirus screening); NK-mSmad7 (NK cells screened for overexpression of lentivirus); KK-control (normal cultured KK cells); KK-NC (KK cells screened against lentivirus); KK-shSmad7 (KK cells that interfere with lentivirus screening); KK-mSmad7 (KK cells screened for overexpression of lentivirus). Cell proliferation was observed by the CCK-8 method, cell apoptosis was detected by flow cytometry, cell migration ability was detected by Transwell chamber, and expression of key proteins (N-cadherin and Occludin) in epithelium-interstitial transform was detected by Western blot. The Smad7 interfering lentivirus vector and Smad7 overexpressing lentivirus vector were successfully constructed. Interference with Smad7 can promote NK cell and KK cell proliferation and migration, and inhibit KK cell apoptosis, but it has no significant effect on NK cell apoptosis ( P>0.05). Overexpression of Smad7 inhibited the proliferation and migration of NK cells and KK cells, and promoted their apoptosis. After interfering with lentivirus infection, NK cells and KK cells showed decreased expression of Occludin protein compared with NC group ( P<0.01), increased N-cadherin protein expression in KK cells ( P<0.01), but there was no significant change in N-cadherin protein expression in NK cells ( P>0.05); After lentivirus overexpression, NK and KK cells showed increased expression of Occludin protein ( P<0.05), the expression of N-cadherin protein in NK cells decreased ( P<0.05), but there was no significant change in N-cadherin protein expression in KK cells ( P>0.05). The regulation of Smad7 gene can affect the EMT in normal skin keratinocytes and keloid keratinocytes, and further regulate the ability of cell proliferation, migration and apoptosis. The effect of Smad7 gene regulation on EMT in keloid keratinocytes was greater than that on normal skin keratinocytes.

The expression of NKT like cells and NK cells in the peripheral blood among patients with idiopathic dilated cardiomyopathy (IDCM)

Abstract Introduction Dilated cardiomyopathy is associated with various immunological abnormalities, such as decreases in the activity and subsets of suppressor T cells and in the activity of natural killer cells, suggesting the involvement of immunological mechanisms in the pathogenesis of this disease. However, the role of NKT like cells and NK cells in the pathogenesis of idiopathic dilated cardiomyopathy remains still uncertain. The aim of the study was to compare the frequencies of peripheral NKT like cells and NK cells among patients with IDCM to healthy controls. Methods The frequencies of NKT like cells and NK cells were measured by flow of cytometry among 50 patients with IDCM from Cardiology Clinic in Lublin. The control group consisted of 50 healthy sex- and age- matched volunteers. The diagnosis of IDCM was based on the common known ESC Guidenlines 2018 criteria. Statistical analysis of the results was conducted using IBM program. A value of p less than 0.05 was considered statistically significant. Results The frequency (%) (mean ± SD) of NK cells (CD3+/CD16+CD56+) was significantly lower (p≤0.001) in IDCM group (12.0±4.5) when compared to control group (15.6±3.4). The frequency (%) (mean ± SD) of NK T like cells (CD3+/CD16+CD56+) was significantly elevated (p≤0,04) in IDCM group (4,66±3,4) in comparison to the control group (3,23±1,7). Conclusions Our findings of the abnormalities in immune cells distribution in peripheral blood of IDCM patients suggest that IDCM development and progression is related to the dysregulation of the immune system. The decreases in the activity in the NK cells among patients with IDCM might indicate chronic myocarditis and lymphocyte infiltration in the myocardium. Moreover, the elevation of NKT like cells could be a result of prolonged exposure to pathogen in which TCD3+ cells become involved more than less mature NK cells in engaging specific immunity response. Funding Acknowledgement Type of funding source: Private hospital(s). Main funding source(s): Medical University in Lublin, Grant for Young Reserchers

Expression of NK cell receptor ligands in primary colorectal cancer tissue in relation to the phenotype of circulating NK- and NKT cells, and clinical outcome

IntroductionNatural killer (NK) cells and natural killer T (NKT) cells are implicated in the development and progression of colorectal cancer (CRC). Tumor cells express NK cell receptor ligands that modulate their function. This study aimed to investigate the expression of such ligands in CRC in relation to the phenotype of circulating NK- and NKT cells, and clinical outcome. MethodsPrimary tumor tissues were analyzed for protein expression of NK cell ligands using immunohistochemistry with automated image analysis in a cohort of 78 CRC patients. For 24 of the 78 patients, RNA expression of NK cell ligands was analyzed in primary tumor tissue using RNA sequencing. Receptor expression on circulating NK- and NKT cells was previously measured by us in 71 of the 78 patients using flow cytometry. ResultsHigh Proliferating Cell Nuclear Antigen (PCNA) protein expression in the primary tumor associated with shorter disease-free survival (DFS) of CRC patients (P = 0.026). A trend was observed towards shorter DFS in CRC patients with above-median galectin-3 protein expression in the primary tumor (P = 0.055). High protein expression of galectin-3, CD1d, and human leukocyte antigen (HLA) class I, and high RNA expression of UL16-binding protein (ULBP)-1, -2, and -5, and HLA-E in the tumor tissue correlated with low expression of the corresponding receptors on circulating NK- or NKT cells (P < 0.05). ConclusionsGalectin-3 and PCNA expression in the primary tumor may be prognostic biomarkers in CRC patients. Furthermore, our results suggest that NK cell receptor ligands expressed by tumor cells may modulate the phenotype of circulating NK- and NKT cells, and facilitate immune escape of metastasizing cells.

Notch signaling defects in NK cells in patients with cancer.

Altered expressions of proto-oncogenes have been reported during normal lymphocytes mitogenesis and in T and B lymphocytes in patients with autoimmune diseases. We have recently demonstrated a significantly decreased expression of c-kit and c-Myc in NK cells isolated from patients with cancer, which might be related to the functional deficiency of NK cells in the tumor environment. Here, focusing on the regulatory mechanisms of this new clinical phenomenon, we determined expression of c-Myc, Notch1, Notch2, p-53, Cdk6, Rb and phosphorylated Rb in NK cells isolated from the healthy donors and cancer patients. The results of our study revealed a significant down-regulation of expression of Notch receptors and up-regulation of Cdk6 expression in NK cells in cancer, while no significant changes in the expression of p53 and Rb proteins were seen. These data revealed novel signaling pathways altered in NK cells in the tumor environment and support further investigation of the origin of deregulated expression of proto-oncogenes in NK cells patients with different types of cancer.

Effects of Dasatinib on the Expansion, Subsets, Receptor Expression and Cytotoxic Function of NK Cells in Vitro

To investigate the effect of dasatinib on the expansion of NK cells in vitro, as well as the subsets, receptor expression and cytotoxic function of NK cells. Peripheral blood mononuclear cells (PBMCs) were isolated from healthy adult volunteers and cultured with SCGM added IL-2 and IL-15 for expansion of NK cells. In this culture system, dasatinib of different concentrations were added. Cell counting and phenotyping by flow cytometry were used to evaluate the amplification efficiency of NK cells. FCM was used to detect the expression of receptors on the surface of NK cells and the distribution of subsets. Subsequently, degranulation assay and CFSE/7AAD based cytotoxicity assay were used to detect the effects of dasatinib on NK cytotoxicity against leukemia cell line K562 cells. The expansion efficiency of NK cells in vitro could be increased by dasatinib at the concentration range of 5-50 nmol/L, and the expansion efficiency of NK cells reached the peak at 20 nmol/L of dasatinib. The NK cytotoxicity against K562 cells in dasatinib cultured group at the concentration of 20 nmol/L was significantly higher than that in control group. For the cells cultured by disatinib in vitro, the MFI of CD226, NKP46 and NKG2D was up-regulated; the ratio of NKG2A+CD57- subset was down-regulated, while the ratio of NKG2A-CD57+ subset was up-regulated.The degranulation response of NKG2A-CD57+ NK cells to K562 cells was stronger than that of NKG2A+CD57- NK cells. The results shows that appropriate dose of dasatinib(20 nmol/L) can increases the amplification efficiency of NK cells, simultaneously up-regulates the expression of NK activating receptors and increases the NKG2A-CD57+ subset, which lead to the enhancement of NK cytotoxicty against leukemia cell lines.

O15 Recognition of subclinical macrophage activation syndrome in an adolescent systemic juvenile idiopathic arthritis patient receiving tocilizumab: a case report

O15 Recognition of subclinical macrophage activation syndrome in an adolescent systemic juvenile idiopathic arthritis patient receiving tocilizumab: a case report

Ascorbic Acid Promotes KIR Demethylation during Early NK Cell Differentiation.

Variegated expression of killer Ig-like receptors (KIR) in human NK cells is a stochastic process exclusive to subsets of mature NK cells and CD8+ T cells. Allele-specific KIR expression is maintained by DNA methylation within the proximal promoter regions. Because KIR genes are densely methylated in NK cell progenitors, there is an implied stage of human NK cell development in which DNA demethylation takes place to allow for active transcription. When and how this process occurs is unknown. In this study, we show that KIR proximal promoters are densely methylated in less mature CD56bright NK cells and are progressively demethylated in CD56dim NK cells as they mature and acquire KIR. We hypothesized that ten-eleven translocation (TET) enzymes, which oxidize 5mC on DNA could mediate KIR promoter demethylation. The catalytic efficiency of TET enzymes is known to be enhanced by ascorbic acid. We found that the addition of ascorbic acid to ex vivo culture of sorted CD56bright NK cells increased the frequency of KIR expression in a dose-dependent manner and facilitated demethylation of proximal promoters. A marked enrichment of the transcription factor Runx3 as well as TET2 and TET3 was observed within proximal KIR promoters in CD56bright NK cells cultured with ascorbic acid. Additionally, overexpression of TET3 and Runx3 promoted KIR expression in CD56bright NK cells and NK-92 cells. Our results show that KIR promoter demethylation can be induced in CD56bright, and this process is facilitated by ascorbic acid.

ABCL-041: Soluble MIC-A and MIC-B, and NKG2D Expression in NK Cells, NKT Cells, and Gamma-Delta T Lymphocytes in Patients with De Novo Non-Hodgkin Lymphoma and Post-Chemotherapy

Context: The presence of soluble NKG2D receptor ligands in different types of cancer affects the cytotoxic functionality of NK cells by interfering with the receptor. Objective: Determine the levels of soluble MIC-A and MIC-B (sMICA, sMICB), the NKG2D receptor and the subpopulations of NK, NKT and Tγδ cells, in patients with de novo NHL and post-chemotherapy, and its relationship to treatment response. Design: Peripheral blood (SP) in EDTA from patients diagnosed with NHL was taken prior to the start of treatment and at least 3 months after chemotherapy. Healthy donors (SD) were included as controls. Setting: By flow cytometry using monoclonal antibodies lymphocyte subpopulations and NKG2D receptor expression were determined, and by ELISA, the levels of sMICA and sMICB. Patients or other participants: 50 patients with de novo NHL, indolent (N=17) and aggressive (N=33), from these 26 post-chemotherapy samples were obtained. Patients of both genders, over 18 years old, with informed consent, never treaty with chemotherapy and without contaminating viral disease. Interventions: There were not, only the sample taken described in setting. Main outcome measures: Before carrying out the project we hypothesized that the high levels of sMICA and sMICB in patients with NHL, as well as the low expression of the NKG2D receptor in SP of these patients are associated with poor response to treatment and poor prognosis in disease evolution. Results: Decrease (P Conclusions: The high level of sMIC in the serum of patients with NHL and the decrease of circulating dendritic, NK, helper (Th), and TCD4reg cells propose a possible mechanism for evasion of the antitumor response of NHL cells.

Abstract 909: The role of checkpoints in Rituximab-mediated NK cell activation

Abstract Purpose: Despite the remarkable clinical success of anti-CD20 monoclonal antibody (mAb) therapy, the mechanisms of action by which such mAb mediate their anti-tumor effects are not completely understood. This includes a need to better understand the role of various NK cell subtypes and checkpoint molecules in anti-CD20 mAb therapy. Experimental procedures: Raji B cell lymphoma cells and peripheral blood mononuclear cells (PBMCs) from normal donors (n=21) were co-cultured with rituximab (RTX) or trastuzumab as a control mAb for up to 15 days. Analysis included assessment of proliferation, remaining viable cells and phenotype of various cell types. Results: As expected, RTX was superior to trastuzumab in eliminating Raji cells. RTX also induced proliferation of NK cells and a shift of NK cells from a CD56dim to CD56bright population. Both NK cell proliferation and the increase in the CD56bright population occurred at day 5-7 when elimination of Raji cells was largely complete. Addback of additional Raji cells at day 5 delayed proliferation of NK cells and the shift from a CD56dim to CD56bright population suggesting this shift occurs once activation of the NK cell via Fcγ;;R is complete. Evaluation of checkpoint molecule expression by NK cells demonstrated RTX, in the presence of Raji target cells, upregulates NK cell expression of TIGIT and TIM3, but not PD1, CTLA-4, or LAG-3. This upregulation was maintained for the full duration of incubation (up to 15 days). The level of expression of TIGIT and TIM3 on NK cells induced by RTX was dose dependent and peaked at 1-5 ug/ml. NK cells induced by RTX to express TIGIT or TIM3 degranulated (CD107a), produced cytokines (IFNγ, TNFα) and expressed activation markers (CD69 and CD25) to a greater degree than did TIGIT or TIM3 negative NK cells. Anti- TIGIT mAb enhanced CD107a expression and TNFα production by RTX-activated NK cells. Conclusion: NK cells proliferate and shift from CD56dim to CD56bright population after being activated and eliminating RTX-coated target cells. TIGIT and TIM3 are biomarkers for RTX-mediated NK cell activation. Blocking TIGIT can enhance RTX-mediated NK cell degranulation and cytokine production. Citation Format: Zhaoming Wang, George J. Weiner. The role of checkpoints in Rituximab-mediated NK cell activation [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 909.

Tumor inhibition or tumor promotion? The duplicity of CXCR3 in cancer.

Tumor tissue includes cancer cells and normal stromal cells such as vascular endothelial cells, connective tissue cells (cancer associated fibroblast, mesenchymal stem cell), and immune cells (tumor-infiltrating lymphocytes or TIL, dendritic cells, eosinophils, basophils, mast cells, tumor-associated macrophages or TAM, myeloid-derived suppressor cells or MDSC). Anti-tumor activity is mainly mediated by infiltration of NK cells, Th1 and CD8+ Tcells, and correlates with expression of NK cell and Tcell attracting chemokines. Nevertheless, cancer cells hijack tissue homeostasis through secretion of cytokines and chemokines that mediate not only the induction of an inflamed status that supports cancer cell survival and growth, but also the recruitment and/or activation of immune suppressive cells. CXCL9, CXCL10, and CXCL11 are known for their tumor-inhibiting properties, but their overexpression in several hematologic and solid tumors correlates with disease severity, suggesting a role in tumor promotion. The dichotomous nature of CXCR3 ligands activity mainly depends on several molecular mechanisms induced by cancer cells themselves able to divert immune responses and to alter the whole local environment. A deep understanding of the nature of such phenomenon may provide a rationale to build up a CXCR3/ligand axis targeting strategy. In this review, we will discuss the role of CXCR3 in cancer progression and in regulation of anti-tumor immune response and immunotherapy.