Effects of organochlorine pesticides on interleukin secretion from lymphocytes

2006 ◽  
Vol 25 (11) ◽  
pp. 651-659 ◽  
Author(s):  
T M Beach ◽  
M M Whalen
Keyword(s):  
T Cells ◽  
Nk Cells ◽  
Organochlorine Pesticides ◽  
Cell Function ◽  
Nk Cell ◽  
Organochlorine Compound ◽  
Organochlorine Compounds ◽  
Negative Effects ◽  
Lytic Function ◽  
Baseline Levels

Organochlorine pesticides have been used worldwide primarily as insecticides. Due to their chemical stability, they often persist in the environment long after their use has ceased. In a previous study, we found that six organochlorine compounds (α-chlordane, γ-chlordane, 4,4′-DDT, heptachlor, oxychlordane, and pentachlorophenol (PCP)), at concentrations of 5 μM, were able to significantly decrease the ability of highly purified human natural killer (NK) cells to lyse tumor cells after exposures, ranging from 1 hour to 6 days. However, if T cells were present with the NK cells (T/NK cells), loss of lytic function was seen only with oxychlordane and PCP. The purpose of the current study is to begin to investigate the mechanism by which T cells may be blocking the negative effects of some organochlorine compounds on NK cell function. Here, we investigated the hypothesis that T cells could produce significant levels of NK-stimulatory interleukin(s) (ILs), and that this may account for the decreased inhibition seen with organochlorine exposures when T cells were present. Secretion of four cytokines that have a demonstrated capacity to influence NK function, and/or are secreted by T cells, was measured (IL-2, IL-4, IL-10, IL-12). We measured both the baseline levels of ILs and the effects of organochlorine compound on IL secretion in T/NK cells. The results showed that baseline levels of the NK-stimulatory IL, IL-12, were 898±264 pg/mL at 24 hours and IL-10 levels were 564±337 pg/mL. In contrast, IL-2 levels were 14±10 pg/mL, and IL-4 levels were 3±2 pg/mL at 24 hours. The two compounds that retained their capacity to decrease NK lytic function in T/NK cells, oxychlordane (5 μM) and PCP (5 and 10 μM), were able to either decrease the secretion of NK-stimulatory ILs (IL-2, IL-12 and/or IL-10) and/or increase secretion of the NK-inhibitory cytokine, IL-4, at each length of exposure tested.

Immunomodulation of human natural killer cell cytotoxic function by organochlorine pesticides

2004 ◽  
Vol 23 (10) ◽  
pp. 463-471 ◽  
Author(s):  
Adrian Reed ◽  
Leticia Dzon ◽  
Bommanna G Loganathan ◽  
Margaret M Whalen
Keyword(s):  
T Cells ◽  
Nk Cells ◽  
Tumor Cells ◽  
Organochlorine Pesticides ◽  
Mononuclear Cells ◽  
Viral Infections ◽  
Nk Cell ◽  
Cell Preparation ◽  
Lytic Function ◽  
Cytotoxic Function

Organochlorine pesticides are used worldwide. To our knowledge there have been no studies dealing with the effects of these agents under in vitro conditions on human natural killer (NK) cell cytotoxic function. NK cells play a central role in immune defense against tumor development and viral infections. Thus, any agent that interferes with the ability of NK cells to lyse their targets could increase the risk of tumor incidence and/or viral infections. In this study, we examined the effects of organochlorine pesticides and some of their breakdown products on the ability of human NK cells to lyse tumor cells. A total of 11 compounds were tested. The compounds were tested in both purified NK cells as well as a cell preparation that contained other mononuclear cells (predominantly T cells) and NK lymphocytes (referred to as T/NK cells). Lymphocytes were exposed to the compounds for periods of time ranging from 1 hour to 6 days. Exposure of highly purified NK cells to 5 μ M α-chlordane, γ-chlordane, 4,4'-DDT, heptachlor, oxychlordane, or pentachlorophenol (PCP) inhibited their ability to destroy K562 tumor-cells by 88±5, 92±8, 61±13%, 64±10%, 69±11%, 76±12%, respectively, after a 24h exposure. The loss of cytotoxic function seen with α-and γ-chlordane remained essentially constant out to 6 days, while that seen with 4,4'-DDT, oxychordane and PCP increased with longer exposures (6 d). PCP was the most effective of the compounds tested at decreasing NK function. Of the compounds that caused decreased lytic function when tested in purified NK cells, only PCP and oxychordane decreased the lytic function of the T/NK cell preparation after any exposure. The results provide evidence of relative toxic potential for the 11 compounds and their immunomodulatory effects on other mononuclear cells (such as T-cells, B-cells, and monocytes) as well as NK lymphocyte function.

scholarly journals Interferon Alpha Enhances NK Cell Function and the Suppressive Capacity of HIV-Specific CD8+T Cells

2018 ◽  
Vol 93 (3) ◽  
Author(s):  
Abena K. R. Kwaa ◽  
Chloe A. G. Talana ◽  
Joel N. Blankson
Keyword(s):  
T Cells ◽  
T Cell ◽  
Nk Cells ◽  
Effector Cell ◽  
Cell Function ◽  
Nk Cell ◽  
Suppressive Capacity ◽  
Short Course ◽  
Shock And Kill ◽  
Hiv 1

ABSTRACTCurrent shock-and-kill strategies for the eradication of the HIV-1 reservoir have resulted in blips of viremia but not in a decrease in the size of the latent reservoir in patients on suppressive antiretroviral therapy (ART). This discrepancy could potentially be explained by an inability of the immune system to kill HIV-1-infected cells following the reversal of latency. Furthermore, some studies have suggested that certain latency-reversing agents (LRAs) may inhibit CD8+T cell and natural killer (NK) cell responses. In this study, we tested the hypothesis that alpha interferon (IFN-α) could improve the function of NK cells from chronic progressors (CP) on ART. We show here that IFN-α treatment enhanced cytokine secretion, polyfunctionality, degranulation, and the cytotoxic potential of NK cells from healthy donors (HD) and CP. We also show that this cytokine enhanced the viral suppressive capacity of NK cells from HD and elite controllers or suppressors. Furthermore, IFN-α enhanced global CP CD8+T cell cytokine responses and the suppressive capacity of ES CD8+T cells. Our data suggest that IFN-α treatment may potentially be used as an immunomodulatory agent in HIV-1 cure strategies.IMPORTANCEData suggest that HIV+individuals unable to control infection fail to do so due to impaired cytokine production and/cytotoxic effector cell function. Consequently, the success of cure agendas such as the shock-and-kill strategy will probably depend on enhancing patient effector cell function. In this regard, NK cells are of particular interest since they complement the function of CD8+T cells. Here, we demonstrate the ability of short-course alpha interferon (IFN-α) treatments to effectively enhance such effector functions in chronic progressor NK cells without inhibiting their general CD8+T cell function. These results point to the possibility of exploring such short-course IFN-α treatments for the enhancement of effector cell function in HIV+patients in future cure strategies.

scholarly journals KIR reconstitution is altered by T cells in the graft and correlates with clinical outcomes after unrelated donor transplantation

Blood ◽  
2005 ◽  
Vol 106 (13) ◽  
pp. 4370-4376 ◽  
Author(s):  
Sarah Cooley ◽  
Valarie McCullar ◽  
Rosanna Wangen ◽  
Tracy L. Bergemann ◽  
Stephen Spellman ◽  
...  
Keyword(s):  
T Cells ◽  
Nk Cells ◽  
Clinical Outcomes ◽  
Hematopoietic Cell Transplantation ◽  
Cell Function ◽  
Nk Cell ◽  
Interferon Γ ◽  
Unrelated Donor ◽  
Ifn Γ

Although unrelated hematopoietic cell transplantation (HCT) is curative for many hematologic malignancies, complications and relapse remain challenging obstacles. Natural killer (NK) cells, which recover quickly after transplantation, produce cytokines and express killer immunoglobulin-like receptors (KIRs) that regulate their cytotoxicity. Some clinical trials based on a KIR ligand mismatch strategy are associated with less relapse and increased survival, but results are mixed. We hypothesized that T cells in the graft may affect NK cell function and KIR expression after unrelated transplantation and that these differences correlate with clinical outcomes. NK cell function was evaluated using 77 paired samples from the National Marrow Donor Program Research Repository. Recipient NK cells at 100 days after both unmanipulated bone marrow (UBM) and T-cell depleted (TCD) transplants were compared with NK cells from their healthy donors. NK cells expressed fewer KIRs and produced more interferon γ (IFN-γ) after UBM compared to TCD transplants. Multivariate models showed that increased NK cell IFN-γ production correlated with more acute graft-versus-host disease (GVHD), and decreased KIR expression correlated with inferior survival. These results support the notion that T cells in the graft affect NK cell reconstitution in vivo. Understanding these mechanisms may result in strategies to improve clinical outcomes from unrelated HCT.

Ifosfamide impairs the allostimulatory capacity of human dendritic cells by intracellular glutathione depletion

Blood ◽  
2003 ◽  
Vol 102 (10) ◽  
pp. 3668-3674 ◽  
Author(s):  
Maria C. Kuppner ◽  
Anabel Scharner ◽  
Valeria Milani ◽  
Christoph von Hesler ◽  
Katharina E. Tschöp ◽  
...  
Keyword(s):  
T Cells ◽  
Dendritic Cells ◽  
T Cell ◽  
Nk Cells ◽  
Cell Function ◽  
Nk Cell ◽  
Human Monocyte ◽  
Glutathione Depletion ◽  
Intracellular Glutathione ◽  
Ifn Γ

AbstractIfosfamide, a clinically potent chemotherapeutic agent, causes the depletion of intracellular glutathione (GSH) levels in various cell types. GSH is the major intracellular reductant against oxidative stress. 4-Hydroxyifosfamide (4-OH-IF), the activated form of ifosfamide, depletes GSH levels in T cells and natural killer (NK) cells; this is accompanied by a decrease in T-cell and NK-cell function. Here we demonstrate for the first time that human monocyte-derived dendritic cells (DCs) express higher constitutive levels of GSH and are less sensitive to 4-OH-IF-induced GSH depletion than T cells and NK cells. Treatment of DCs with 4-OH-IF significantly reduced their ability to stimulate allogeneic T-cell proliferation and interferon-γ (IFN-γ) production. Ifosfamide also decreased DC interleukin-12p70 (IL-12p70) production after stimulation with lipopolysaccharide (LPS) and IFN-γ. The decrease in allostimulatory capacity and in IFN-γ and IL-12 production correlated with a decrease in intracellular GSH in the DCs. The responses could be restored by reconstituting DC GSH levels with glutathione monoethyl ester (GSH-OEt). 4-OH-IF had no inhibitory effect on the ability of DCs to present exogenously added tyrosinase peptide to tyrosinase-specific cytotoxic T lymphocytes (CTLs). These studies suggest that in cancer patients treated with ifosfamide, protection strategies based on glutathione reconstitution may enhance DC function. (Blood. 2003;102: 3668-3674)

scholarly journals 216 Multiplex base editing of NK cell to enhance cancer immunotherapy

2021 ◽  
Vol 9 (Suppl 3) ◽  
pp. A229-A229
Author(s):  
Minjing Wang ◽  
Mitchell Kluesner ◽  
Patricia Claudio Vázquez ◽  
Beau Webber ◽  
Branden Moriarity
Keyword(s):  
T Cells ◽  
Nk Cells ◽  
Cancer Immunotherapy ◽  
High Efficiency ◽  
Cell Function ◽  
Nk Cell ◽  
Functional Assay ◽  
Single Base ◽  
Editing Efficiency ◽  
Base Editing

BackgroundNatural killer (NK) cells have many unique features that have gained attention in cancer immunotherapy. NK cells can kill in antigen independent and dependent fashion, can be used as an allogeneic product, and perform antibody-dependent cell-mediated cytotoxicity (ADCC). However, NK cell function is regulated by many activating and inhibitory receptors, which cancer cells take advantage of to avoid being killed by NK cells. NK cells are also known for their technical and biological challenges which result in low editing efficiencies, compared to T cells and other immune cells.MethodsBase editing (BE) is a CRISPR-Cas9 based genome editing technology that allows precise single base transitions. Previously, we reported a high efficiency method for multiplex engineering of T cells using BE and thus reasoned that applying similar concepts in NK cells may offer an opportunity to alter many genes simultaneously at higher efficiency through multiplex base editing. We thus selected a panel of genes bearing critical roles in NK cell function for immunotherapy, including inhibitory intracellular regulator AHR and CISH, inhibitory checkpoint receptor KLRG1, TIGIT, KLRC1, and PDCD1, and Fc receptor CD16A. CD16A is responsible for NK cell ADCC and is regulated via cleavage upon NK activation. Non-cleavable CD16A improves ADCC killing and can be achieved through single-base substitution with BE.ResultsUsing the adenosine BE (ABE8e), we achieved multiplex editing (6 genes) rates up to 99% and 95% editing/knockout at DNA and protein levels, respectively. Notably, we assessed for reduction in editing efficiency when additional genes were targeted and found no significant reduction in editing efficiencies when targeting up to 6 genes simultaneously. Moreover, functional evaluation of non-cleavable CD16A NK cells revealed up to 35% increase of cytotoxicity against Raji cells.ConclusionsWe were able to achieve high multiplex editing efficiency in primary human NK cells using ABE8eand there was no significant decrease of editing efficiency as the number of gene of interest increases, up to 6 genes in total. Functional assay confirmed increased NK cell cytotoxicity against tumor cells. Our end goal is to achieve high efficiency multiplex editing in CAR-expressing NK cells to further improve NK cell activity and toxicity for cancer immunotherapy.ReferenceWebber B, Lonetree C, Kluesner M, et al. Highly efficient multiplex human T cell engineering without double-strand breaks usingCas9 base editors. Nat Commun 2019;10:5222.

scholarly journals Acute rejection of murine bone marrow allografts by natural killer cells and T cells. Differences in kinetics and target antigens recognized.

1987 ◽  
Vol 166 (5) ◽  
pp. 1499-1509 ◽  
Author(s):  
W J Murphy ◽  
V Kumar ◽  
M Bennett
Keyword(s):  
Bone Marrow ◽  
T Cells ◽  
Nk Cells ◽  
Nude Mice ◽  
Cell Function ◽  
Nk Cell ◽  
Marrow Cell ◽  
Severe Combined Immunodeficiency ◽  
Allogeneic Bone ◽  
Murine Bone Marrow

Lethally irradiated C.B-17 +/+, C.B-17 scid/scid (severe combined immunodeficiency, SCID), BALB/c-nu/nu (nude), and C57BL/6 (B6) mice were challenged with H-2-homozygous or H-2-heterozygous totally allogeneic bone marrow cell (BMC) grafts. Some of the irradiated mice were immunized simultaneously with large numbers of irradiated marrow and spleen cells syngeneic with the viable BMC transferred. Irradiated SCID and nude mice, devoid of T cells but with normal NK cell function, were able to reject H-2-homozygous BMC grafts within 4 d. However, they were unable to reject H-2-heterozygous BMC allografts by 7 d even if they were immunized. B6 and C.B-17 +/+ mice were able to reject H-2 heterozygous BMC allografts by 7-8 d, but not as early as 4 d, if they were immunized. The rejection of H-2-homozygous BMC on day 4 was inhibited by administration of anti-NK-1.1 antibodies, but not by anti-Lyt-2 antibodies. Conversely, the rejection of H-2-heterozygous allogeneic BMC on day 8 was prevented by anti-Lyt-2 but not by anti-NK-1.1 antibodies. The data indicate that both NK cells and Lyt-2+ T cells can mediate rejection of allogeneic BMC acutely, even after exposure of mice to lethal doses of ionizing irradiation. NK cells appear to recognize Hemopoietic histocompatibility (Hh) antigens on H-2 homozygous stem cells. The inability of SCID and nude mice to reject H-2 heterozygous totally allogeneic BMC indicate that NK cells do not survey donor marrow cells for self H-2 antigens and reject those cells that express nonself H-2 antigens. The T cells presumably recognize conventional H-2 antigens (probably class I) under these conditions.

scholarly journals Blockade of checkpoint receptor PVRIG unleashes anti-tumor immunity of NK cells in murine and human solid tumors

2021 ◽  
Vol 14 (1) ◽  
Author(s):  
Yangyang Li ◽  
Yu Zhang ◽  
Guoshuai Cao ◽  
Xiaodong Zheng ◽  
Cheng Sun ◽  
...  
Keyword(s):  
Colon Cancer ◽  
T Cells ◽  
Tumor Growth ◽  
Nk Cells ◽  
Cell Function ◽  
Nk Cell ◽  
Therapeutic Potential ◽  
Human Tumor ◽  
Murine Tumor

Abstract Background Although checkpoint-based immunotherapy has shown exciting results in the treatment of tumors, around 70% of patients have experienced unresponsiveness. PVRIG is a recently identified immune checkpoint receptor and blockade of which could reverse T cell exhaustion to treat murine tumor; however, its therapeutic potential via NK cells in mice and human remains seldom reported. Methods In this study, we used patient paraffin-embedded colon adenocarcinoma sections, various murine tumor models (MC38 colon cancer, MCA205 fibrosarcoma and LLC lung cancer), and human NK cell- or PBMC-reconstituted xenograft models (SW620 colon cancer) to investigate the effect of PVRIG on tumor progression. Results We found that PVRIG was highly expressed on tumor-infiltrating NK cells with exhausted phenotype. Furthermore, either PVRIG deficiency, early blockade or late blockade of PVRIG slowed tumor growth and prolonged survival of tumor-bearing mice by inhibiting exhaustion of NK cells as well as CD8+ T cells. Combined blockade of PVRIG and PD-L1 showed better effect in controlling tumor growth than using either one alone. Depletion of NK or/and CD8+ T cells in vivo showed that both cell types contributed to the anti-tumor efficacy of PVRIG blockade. By using Rag1−/− mice, we demonstrated that PVRIG blockade could provide therapeutic effect in the absence of adaptive immunity. Further, blockade of human PVRIG with monoclonal antibody enhanced human NK cell function and inhibited human tumor growth in NK cell- or PBMC-reconstituted xenograft mice. Conclusions Our results reveal the importance of NK cells and provide novel knowledge for clinical application of PVRIG-targeted drugs in future.

scholarly journals Distinct immune composition in lymph node and peripheral blood of CLL patients is reshaped during venetoclax treatment

2019 ◽  
Vol 3 (17) ◽  
pp. 2642-2652 ◽  
Author(s):  
Iris de Weerdt ◽  
Tom Hofland ◽  
Renate de Boer ◽  
Johan A. Dobber ◽  
Julie Dubois ◽  
...  
Keyword(s):  
T Cells ◽  
Lymph Node ◽  
Nk Cells ◽  
Peripheral Blood ◽  
Cell Function ◽  
Nk Cell ◽  
B Cell Lymphoma ◽  
Lymphocytic Leukemia ◽  
Leukemic Cells ◽  
Cytotoxic Lymphocytes

Abstract Morbidity and mortality due to immunosuppression remain among the foremost clinical challenges in chronic lymphocytic leukemia (CLL). Although immunosuppression is considered to originate within the lymph node (LN) microenvironment, alterations in T and natural killer (NK) cells have almost exclusively been studied in peripheral blood (PB). Whereas chemoimmunotherapy further deteriorates immune function, novel targeted agents like the B-cell lymphoma 2 inhibitor venetoclax potentially spare nonmalignant lymphocytes; however, the effects of venetoclax on nonleukemic cells have not been explored. We address these unresolved issues using a comprehensive analysis of nonmalignant lymphocytes in paired LN and PB samples from untreated CLL patients, and by analyzing the effects of venetoclax-based treatment regimens on the immune system in PB samples from previously untreated and relapsed/refractory patients. CLL-derived LNs contained twice the amount of suppressive regulatory T cells (Tregs) and CLL supportive follicular T helper (Tfh) cells compared with PB. This was accompanied by a low frequency of cytotoxic lymphocytes. The expression of PD-1 by CD8+ T cells was significantly higher in LN compared with PB. Venetoclax-based treatment led to deep responses in the majority of patients, but also to decreased absolute numbers of B, T, and NK cells. Tfh cell, Treg, and PD-1+ CD8+ T cell numbers were reduced more than fivefold after venetoclax-based therapy, and overproduction of inflammatory cytokines was reduced. Furthermore, we observed restoration of NK cell function. These data support the notion that the immunosuppressive state in CLL is more prominent within the LN. Venetoclax-based regimens reduced the immunosuppressive footprint of CLL, suggesting immune recovery after the elimination of leukemic cells.

scholarly journals Natural Killer Cell Dysfunction in Chronic Lymphocytic Leukemia Is Associated with Loss of the Mature KIR3DL1+ Subset

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 3318-3318 ◽  
Author(s):  
Alexander W. MacFarlane ◽  
Mowafaq Jillab ◽  
Mitchell R Smith ◽  
R. Katherine Alpaugh ◽  
Marion E. Cole ◽  
...  
Keyword(s):  
T Cells ◽  
B Cells ◽  
Chronic Lymphocytic Leukemia ◽  
Nk Cells ◽  
Peripheral Blood ◽  
Cell Function ◽  
Nk Cell ◽  
Tumor Burden ◽  
Lymphocytic Leukemia ◽  
Healthy Controls

Abstract Background: B-cell chronic lymphocytic leukemia (CLL) is a common blood cancer characterized by high prevalence of malignant B cells in peripheral blood. Small lymphocytic lymphoma (SLL) is considered to be a different presentation of the same disease, with the malignant B cells primarily localized in lymph nodes. Natural killer (NK) cells are innate immune effectors that can spontaneously identify and kill malignant cells, especially hematopoietic cancers. In peripheral blood of CLL patients, NK cells are chronically exposed to significant tumor burden, which is predicted to influence their phenotype and function. Effective NK cell function may be particularly beneficial in CLL patients, since commonly-used monoclonal antibody therapies (e.g. rituximab, alemtuzumab) rely at least partially on ADCC-mediated by NK cells. Methods: We performed a prospective analysis of biomarkers on fresh peripheral blood lymphocytes from 25 untreated CLL patients, 10 untreated SLL and 17 age-matched healthy controls by 10-color flow cytometry. All subjects signed IRB approved informed consent forms. Our study analyzed 180 distinct biomarker parameters, with a particular focus on NK and T cells. Differences in biomarker expression between patients with SLL, CLL, and healthy controls were compared by Wilcoxon rank-sum test. Results: Absolute numbers of NK and T cells per µl of blood were significantly higher in CLL patients, and this correlated with increased B cell numbers. As indicators of immune suppression, the frequency of regulatory T cells was significantly increased in CLL samples, as were levels of PD-1 expression on T cells and CD56dim NK cells. NK cells in CLL expressed higher levels of CD27, which is characteristic of a less mature phenotype, and CD56dim cells expressed lower levels of NKG2D. Compared to healthy controls, CLL samples displayed a marked reduction in degranulation by CD56dim NK cells in response to transformed 721.221 B cells, either with or without rituximab. CD56dim NK cells from CLL patients were also less viable under resting conditions or when challenged with target cells, especially in ADCC responses. We further observed a striking reduction in the frequency and viability of KIR3DL1+ NK cells, which progressed over time in most CLL patients. Surprisingly, CLL patients with the highest levels of PD-1 expression on NK cells possessed genes for both KIR3DL1 and its ligand, HLA-Bw4. Our findings were also clearly evident in a CLL patient compared to her healthy monozygotic twin, thereby providing compelling support for the results in the full patient cohort. The altered expression levels of nearly all of the NK cell biomarkers and degranulation were less pronounced in blood samples from SLL patients, presumably due to low tumor burden in peripheral blood. Conclusions: CLL patients have increased numbers of NK cells in peripheral blood, but these NK cells are less mature, are significantly depleted of the KIR3DL1+ subset, and have deficits in degranulation response, reduced expression of NKG2D activating receptor, increased expression of inhibitory PD-1, and enhanced susceptibility to activation-induced death when challenged with tumor targets and rituxumab. Our findings support the hypothesis that immune dysfunction in CLL may be due in part to a selective loss of mature KIR3DL1+ NK cells, possibly upon encountering overwhelming tumor burden in peripheral blood, and CLL patients may benefit from therapeutic strategies that augment NK cell function. Disclosures No relevant conflicts of interest to declare.

599 New checkpoints controlling function of cytotoxic lymphocytes infiltrating human carcinoma

2020 ◽  
Vol 8 (Suppl 3) ◽  
pp. A634-A634
Author(s):  
Anna Herbstritt ◽  
Elfriede Noessner ◽  
Petra Prinz ◽  
Mani Kadiyala ◽  
Melissa Maxwell ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cell Growth ◽  
Tumor Cell ◽  
Nk Cells ◽  
Cell Function ◽  
Nk Cell ◽  
Diacylglycerol Kinase ◽  
Tumor Control ◽  
T Cell Function

BackgroundAlthough present in high numbers, T and NK cells appear functionally impaired in the renal cell carcinoma (RCC) tumor milieu, as they cannot be stimulated to degranulation and IFN-γ production. This is in part due to altered regulation of signaling downstream of the T cell receptor (TCR). Increased diacylglycerol kinase alpha (DGK-α) has been observed in T and NK cells from the RCC tumor microenvironment (TME). Ex vivo inhibition of DGK-α by the commercially available inhibitor R59022 was able to restore responsiveness to stimulation.1 2 Inhibition of DGK-α is reported to also block tumor cell growth and survival.3 4 Many T cells from RCC additionally express the immune checkpoint Programmed cell Death-1 (PD-1). Interaction of PD-1 with PD-L1 on tumor cells blocks AKT signaling and inhibits T cell function. In the clinic, blocking the PD-1/PD-L1 interaction allows tumor control in some patients; however, the majority of patients do not respond long-term. Since DGK-α acts downstream of PD-1 it may, if overactive, curb T cell function despite PD-1/PD-L1 blockade. Thus, we hypothesize that dual inhibition of PD-1 and DGK α might be required to fully unleash the T cell’s potential in the TME.Current DGK-α inhibitors are not suitable for clinical application. Therefore, we investigate alternative means using RNA interference (RNAi) to target DGK-α alone as well as in combination with PD-1.MethodsKnockdown was achieved by RNAi using INTASYLTM compounds, developed by Phio Pharmaceuticals. These compounds incorporate drug-like properties into siRNA, resulting in enhanced uptake with no need for transfection reagents. Efficacy was analyzed on mRNA and protein level by rt-qPCR, flow cytometry and Western Blot. Functional assays include cytotoxicity and cytokine production in tumor-mimicking environments.ResultsUsing INTASYLTM compounds, silencing of DGK-α was observed in human U2OS osteosarcoma as well as K562 erythroleukemic cells. PD-1 knockdown was achieved in human T cells isolated from peripheral blood mononuclear cells (PBMC). Synergy of DGK-α and PD-1 knockdown is tested in tumor-mimicking in vitro systems using T cell/tumor cell co-cultures at high tumor cell density where T and NK cells become functional suppressed as observed in the TME.ConclusionsStrong activity of specific T and NK cells is necessary for tumor control. Dual targeting of PD-1 and DGK-α may be required to fully enable T and NK cell reactivity in the TME. Self-delivering RNAi technology represents a promising approach to targeting intracellular immune checkpoints such as DGK-α, in addition to PD-1 inhibition.ReferencesPrinz PU, Mendler AN, Masouris I, Durner L, Oberneder R, Noessner E. High DGK-α and disabled MAPK pathways cause dysfunction of human tumor-infiltrating CD8+ T cells that is reversible by pharmacologic intervention. J Immunol 2012 Jun 15;188(12):5990–6000. doi: 10.4049/jimmunol.1103028. Epub 2012 May 9. PMID: 22573804.Prinz PU, Mendler AN, Brech D, Masouris I, Oberneder R, Noessner E. NK-cell dysfunction in human renal carcinoma reveals diacylglycerol kinase as key regulator and target for therapeutic intervention. Int J Cancer 2014 Oct 15;135(8):1832–41. doi: 10.1002/ijc.28837. Epub 2014 Mar 26. PMID: 24615391.Torres-Ayuso P, Daza-Martín M, Martín-Pérez J, Ávila-Flores A, Mérida I. Diacylglycerol kinase α promotes 3D cancer cell growth and limits drug sensitivity through functional interaction with Src. Oncotarget 2014 Oct 30;5(20):9710–26. doi: 10.18632/oncotarget.2344. PMID: 25339152; PMCID: PMC4259432.Yanagisawa K, Yasuda S, Kai M, Imai S, Yamada K, Yamashita T, Jimbow K, Kanoh H, Sakane F. Diacylglycerol kinase alpha suppresses tumor necrosis factor-alpha-induced apoptosis of human melanoma cells through NF-kappaB activation. Biochim Biophys Acta 2007 Apr; 1771(4):462–74. doi: 10.1016/j.bbalip.2006.12.008. Epub 2007 Jan 8. PMID: 17276726.

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