scholarly journals TGFβ in the obese liver mediates conversion of NK cells to a less cytotoxic ILC1-like phenotype

2019 ◽  
Author(s):  
Antonia O. Cuff ◽  
Francesca Sillito ◽  
Simone Dertschnig ◽  
Andrew Hall ◽  
Tu Vinh Luong ◽  
...  
Keyword(s):  
Nk Cells ◽  
Nk Cell ◽  
Target Cells ◽  
Alcoholic Fatty Liver ◽  
Associated Proteins ◽  
Profile Characteristic ◽  
The Cost ◽  
Increased Susceptibility

The liver contains both NK cells and their less cytotoxic relatives, ILC1. Here, we investigate the role of NK cells and ILC1 in the obesity-associated condition, non-alcoholic fatty liver disease (NAFLD). In the livers of mice suffering from NAFLD, NK cells are less able to degranulate, express lower levels of perforin and are less able to kill cancerous target cells than those from healthy animals. This is associated with a decreased ability to kill cancer cells in vivo. On the other hand, we find that perforin-deficient mice suffer from less severe NAFLD, suggesting that this reduction in NK cell cytotoxicity may be protective in the obese liver, albeit at the cost of increased susceptibility to cancer. The decrease in cytotoxicity is associated with a shift towards a transcriptional profile characteristic of ILC1, increased expression of the ILC1-associated proteins CD200R1 and CD49a, and an altered metabolic profile mimicking that of ILC1. We show that the conversion of NK cells to this less cytotoxic phenotype is at least partially mediated by TGFβ, which is expressed at high levels in the obese liver. Finally, we show that reduced cytotoxicity is also a feature of NK cells in the livers of human NAFLD patients.

scholarly journals GSK-3α Inhibition in Drug-Resistant CML Cells Promotes Susceptibility to NK Cell-Mediated Lysis in an NKG2D- and NKp30-Dependent Manner

Cancers ◽  
2021 ◽  
Vol 13 (8) ◽  
pp. 1802
Author(s):  
Nayoung Kim ◽  
Mi Yeon Kim ◽  
Woo Seon Choi ◽  
Eunbi Yi ◽  
Hyo Jung Lee ◽  
...  
Keyword(s):  
Nk Cells ◽  
Nk Cell ◽  
Negative Regulator ◽  
Target Cells ◽  
Dependent Manner ◽  
Cell Functions ◽  
Cell Based Therapy ◽  
Activating Receptors ◽  
Gsk 3Β

Natural killer (NK) cells are innate cytotoxic lymphocytes that provide early protection against cancer. NK cell cytotoxicity against cancer cells is triggered by multiple activating receptors that recognize specific ligands expressed on target cells. We previously demonstrated that glycogen synthase kinase (GSK)-3β, but not GSK-3α, is a negative regulator of NK cell functions via diverse activating receptors, including NKG2D and NKp30. However, the role of GSK-3 isoforms in the regulation of specific ligands on target cells is poorly understood, which remains a challenge limiting GSK-3 targeting for NK cell-based therapy. Here, we demonstrate that GSK-3α rather than GSK-3β is the primary isoform restraining the expression of NKG2D ligands, particularly ULBP2/5/6, on tumor cells, thereby regulating their susceptibility to NK cells. GSK-3α also regulated the expression of the NKp30 ligand B7-H6, but not the DNAM-1 ligands PVR or nectin-2. This regulation occurred independently of BCR-ABL1 mutation that confers tyrosine kinase inhibitor (TKI) resistance. Mechanistically, an increase in PI3K/Akt signaling in concert with c-Myc was required for ligand upregulation in response to GSK-3α inhibition. Importantly, GSK-3α inhibition improved cancer surveillance by human NK cells in vivo. Collectively, our results highlight the distinct role of GSK-3 isoforms in the regulation of NK cell reactivity against target cells and suggest that GSK-3α modulation could be used to enhance tumor cell susceptibility to NK cells in an NKG2D- and NKp30-dependent manner.

scholarly journals Soluble and Exosome-Bound α-Galactosylceramide Mediate Preferential Proliferation of Educated NK Cells with Increased Anti-Tumor Capacity

Cancers ◽  
2021 ◽  
Vol 13 (2) ◽  
pp. 298
Author(s):  
Arnika K. Wagner ◽  
Ulf Gehrmann ◽  
Stefanie Hiltbrunner ◽  
Valentina Carannante ◽  
Thuy T. Luu ◽  
...  
Keyword(s):  
Nk Cells ◽  
Cancer Immunotherapy ◽  
Nk Cell ◽  
Mouse Strains ◽  
Target Cells ◽  
Class I Mhc ◽  
Mhc I ◽  
Cell Responses ◽  
Missing Self

Natural killer (NK) cells can kill target cells via the recognition of stress molecules and down-regulation of major histocompatibility complex class I (MHC-I). Some NK cells are educated to recognize and kill cells that have lost their MHC-I expression, e.g., tumor or virus-infected cells. A desired property of cancer immunotherapy is, therefore, to activate educated NK cells during anti-tumor responses in vivo. We here analyze NK cell responses to α-galactosylceramide (αGC), a potent activator of invariant NKT (iNKT) cells, or to exosomes loaded with αGC. In mouse strains which express different MHC-I alleles using an extended NK cell flow cytometry panel, we show that αGC induces a biased NK cell proliferation of educated NK cells. Importantly, iNKT cell-induced activation of NK cells selectively increased in vivo missing self-responses, leading to more effective rejection of tumor cells. Exosomes from antigen-presenting cells are attractive anti-cancer therapy tools as they may induce both innate and adaptive immune responses, thereby addressing the hurdle of tumor heterogeneity. Adding αGC to antigen-loaded dendritic-cell-derived exosomes also led to an increase in missing self-responses in addition to boosted T and B cell responses. This study manifests αGC as an attractive adjuvant in cancer immunotherapy, as it increases the functional capacity of educated NK cells and enhances the innate, missing self-based antitumor response.

Effective Alloreactive NK Cell Therapy for Breast Cancer In Mice Requires Conditioning with Cyclophosphamide and Total Body Irradiation but Not Bone Marrow Engraftment

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 2085-2085
Author(s):  
Michel Van Gelder ◽  
Peter Frings ◽  
Catarina Matos ◽  
Harry C. Schouten ◽  
Gerard M.J. Bos
Keyword(s):  
Breast Cancer ◽  
Bone Marrow ◽  
Nk Cells ◽  
Nk Cell ◽  
Spleen Cells ◽  
Slow Cycling ◽  
4T1 Breast Cancer ◽  
Bone Marrow Engraftment

Abstract Abstract 2085 Background: Patients with metastasized breast cancer cannot be cured by current standard treatment options. One hypothesis is that slow cycling chemo-resistant tumor stem cells give rise to new tumors after cytoreductive treatment, ultimately leading to chemoresistant tumors. Last year we showed that the 4T1 mouse breast cancer model contains slow-cycling chemo-resistant cells that induce renewed growth of the tumor after chemo- and radiotherapy (abstract 4082). We also showed that haploidentical spleen and bone marrow transplantation (BMT) cures the mice and that donor NK cells are a prerequisite. Our current aim was to study the need of long term BM engraftment and to study the role of the conditioning in the curative process. Methods: The 4T1 breast cancer cell line, originating from a spontaneous Balb/c (H-2d) breast cancer, was cultured under standard conditions. Fifty thousand 4T1 cells were injected s.c. in the flank. For the experiments addressing the need for haploidentical BMT tumor bearing CB6F1 (H-2b/d) recipients were treated with 2x 2Gy total body irradiation and 200 mg/kg cyclophosphamide (CY+TBI) followed by in vitro NK cell enriched haploidentical B6CBAF1 (H-2b/k) spleen cell infusion with or without additional BM cells. Chimerism in tumor-free surviving recipients was measured by flowcytometry of spleens at least 100 days after the treatment. The role of the conditioning in the alloreactive NK cell effect was studied in fully H-2 mismatched B6CBAF1 mice. When indicated, in vivo NK cell depletion was by i.p. injection of anti-AsialoGM1. Results: Figure A shows overall survival of mice with breast cancer after various treatments (10 mice per group). Haploidentical BMT plus spleen cells cured 50% of tumor bearing mice after CY+TBI (♦, dashed line) and survival was at least as good when NK cell enriched spleen cells were co-transplanted (▴, solid line). Transplantation of spleen cells from NK cell depleted mice (•, dotted line) obliterated the beneficial effect of haploidentical transplantation and resulted is similar poor survival as syngeneic BMT plus spleen cells (▪, solid line). The majority of mice that received NK cell enriched spleen cells (10 out of 14 tested) had no bone marrow engraftment and in the other four only 1–5% donor cells were detectable at 150 days. Recipients of unmanipulated haploidentical spleen and BM cells had >90% donor chimerism in 10 out of 14 tested. The cure rate in both groups was nevertheless similarly high. In a subsequent experiment (Figure B, 10 mice per group) we infused haploidentical NK cells only after CY+TBI (▴, solid line); other groups received T cell depleted (x, solid line) or T cell replete (♦, solid line) haploidentical BMT, or syngeneic BMT (▪, solid line). This resulted in a similar superior tumor-free survival (80-90%) than in mice co-transplanted with haploidentical BM (90%), as compared with syngeneic BM and spleen cell transplantation (•, dotted line). We then planned to study the role of the conditioning in the curative process. For this purpose 4T1 breast cancer cells were injected in fully H-2 mismatched B6CBAF1 mice (H-2b/k). Surprisingly, 4T1 breast cancer is not rejected by B6CBAF1 mice despite the full MHC mismatch. Tumors are only rejected when the mice were treated with CY+TBI. Tumor rejection proved to be NK cell dependant and not a direct result of the conditioning as it was prevented by in vivo NK cell depletion. Conclusions: This report provides the first evidence that chemo resistant tumor cells can be eliminated in vivo by alloreactive NK cells resulting in cure without the need for long term donor bone marrow engraftment. Conditioning with CY+TBI seems essential for this effect. These results set the stage for the exploration of alloreactive NK therapy in patients with metastasized breast cancer. Disclosures: No relevant conflicts of interest to declare.

Novel monoclonal antibody therapeutics for metastatic castration resistant prostate cancer.

2019 ◽  
Vol 37 (15_suppl) ◽  
pp. e14222-e14222
Author(s):  
Maloy Ghosh ◽  
Kavitha Iyer Rodrigues ◽  
Sunit Maity ◽  
Sanghamitra Bhattacharjee ◽  
Yogendra Manjunath ◽  
...  
Keyword(s):  
Prostate Cancer ◽  
Immune Responses ◽  
Nk Cells ◽  
Nk Cell ◽  
Therapeutic Potential ◽  
Target Cells ◽  
Castration Resistant Prostate Cancer ◽  
Growth Reduction ◽  
Castration Resistant

e14222 Background: Therapeutic potential of innate immunity comprising Natural killer cell based targets are beginning to unravel the complexity of immune responses. NK cells recognize and induce cytotoxicity of wide range of target cells, such as, tumor cells without prior antigen sensitization. In this study, we have studied Lectin-like transcript 1 (LLT1), a member of the C-type lectin super family, is expressed on target cells and various immune cells. LLT1 isoform 1, is known to interact with CD161, a critical receptor on NK cells. CD161 is expressed on most of human NK cells, NK-T cells, γδ T cells and so on. Tumors exploit the CD161- LLT1 interaction to evade host defense mechanism (“DO NOT KILL” signal); indicating LLT1 as an attractive immunotherapeutic strategy. Methods: Prostate cancer cell lines and other tumor cell lines were used to evaluate novel anti LLT1 antibodies for therapeutic potential - IFNγ production assays and tumor cell death assays were carried out. In vivo efficacy of these antibodies were established using PC3 xenograft in humanized mouse model (HuNOG-EXL). Results: Human androgen independent prostate cancer cell line, PC3 was studied for LLT1 expression and interactions with immune cells, to understand role of LLT1 in metastatic castration resistant prostate cancer (mCRPC). Overexpression of LLT1 on tumor cells was influenced by cytokines and various TLRs. Inhibition of CD161-LLT1 interaction with novel anti LLT1 antibodies leads to IFNγ production and consequent NK cell mediated cytotoxicity – hall mark of anti-tumor responses. Disruption of LLT1 - CD161 innate immunity axis with anti LLT1 antibody releases the break on NK cell cytotoxicity and hence, established a new therapeutic option. PC3 xenograft on HuNOG mouse revealed in vivo efficacy of LLT1 antibody. Significant tumor growth reduction was observed with specific anti LLT1 antibodies alone and in combination with check point antibodies. Thus, synergistic tumor growth reduction was established by combinatorial application of anti LLT1 antibody and PD1/PDL1 axis inhibitors. Conclusions: PC3 xenograft study and other results point to therapeutic opportunities in metastatic castration resistant prostate cancer, a disease condition which needs improved patient outcomes. The ligation of CD161/LLT1 will serve as a new immuno-oncology pair regulating innate and adaptive immune responses; novel human antibodies against LLT1 described here will bring therapeutic benefit to patients in need.

scholarly journals Engineered Interleukin-15 Autocrine Signaling Invigorates Anti-CD123 CAR-NK Cells

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 2806-2806
Author(s):  
Ilias Christodoulou ◽  
Michael Koldobskiy ◽  
Won Jin Ho ◽  
Andrew Marple ◽  
Wesley J. Ravich ◽  
...  
Keyword(s):  
Nk Cells ◽  
Peripheral Blood ◽  
Research Funding ◽  
Nk Cell ◽  
Xenograft Model ◽  
Blood Analysis ◽  
Target Cells ◽  
Interleukin 15 ◽  
Peripheral Blood Analysis

Abstract Introduction : Acute Myeloid Leukemia (AML) is an aggressive neoplastic disorder with poor outcomes in children and adults. NK cell adoptive transfer is an anti-cancer immunotherapy that has promise for AML treatment. We aimed to improve NK cell anti-tumor efficacy with expression of a Chimeric Antigen Receptor (CAR) on the cell surface. Our CAR consists of an extracellular single-chain variable fragment targeting the AML-associated antigen CD123 (IL3Rα) and intracellular domains derived from 2B4 and TCRζ. We sought to improve the persistence and long-term functionality of our CAR-NKs by introducing transgenic interleukin-15 (IL15). Methods: CD3-depleted PBMCs were first activated with lethally irradiated feeder cells, then transduced with transiently produced replication incompetent γ-retrovirus (αCD123.2B4.ζ, αCD123.2B4.ζ-IRES-sIL15, sIL15-IRES-mOrange) on day 4 of culture. CAR expression was measured on day 8 using FACS. Secretion of IL15 was verified with ELISA. Cytotoxicity was measured using ffLuc expressing target cells and bioluminescence (BL) measurement. In serial stimulation assays, target cells were repleted daily to maintain a 1:1 effector:target ratio. Immunophenotype and cell counts were assessed by FACS. Transcriptomic analysis (RNAseq) was performed on RNA derived from NK cells purified on D10. Xenograft modeling was performed using NSG mice engrafted with MV-4-11.ffLuc or MOLM-13.ffLuc AML cell lines. Mice were treated with NK cells on D4 or D4-7-10. Untreated mice served as controls. Tumor growth was serially tracked in vivo using BL imaging. NK cell persistence and expansion were measured in peripheral blood. Results: The 2B4.ζ CAR was well expressed on the surface of transduced NK cells (median transduction efficiency 95%, range 85-97%, n=3). 2B4.ζ CAR-NK treatment prolonged survival of AML engrafted mice when compared to treatment with unmodified NKs (median survival: 63 vs 55 days; n=8 mice; p=0.014). Serial peripheral blood analysis revealed a steady decline in circulating NK cells, which were undetectable in all cohorts within 21 days. NK cells were then engineered for constitutive secretion of IL15, with and without CAR expression. 2B4.ζ/sIL15 CAR-NKs had the most potent 24h-cytotoxicity against CD123+ targets (Fig. 1). After a 10-day chronic stimulation with MV-4-11, 2B4.ζ/sIL15- and sIL15-NKs expanded (x1.2 and x5.9 respectively), while NK cells without sIL15 decreased in number. In this assay, only 2B4.ζ/sIL15 CAR-NKs exhibited sustained tumor killing. Transcriptomic analysis after 10 days of serial stimulation showed sample clustering dependent on IL15 secretion. Differential gene expression analysis (DESeq2) identified upregulation of genes associated with cell cycle progression, apoptosis regulation, chemokine signaling, and NK cell mediated cytotoxicity in NK cells secreting IL15 compared to those without. In multiparameter flow cytometric analysis, 2B4.ζ/sIL15 CAR-NKs had a higher percentage of NK cells populating clusters defined by higher surface expression of NK cell activating receptors (NKp30, NKG2D, LFA-1) compared to 2B4.ζ and unmodified NK cells. In our MV-4-11 xenograft model, NKs armed with secreted IL15 expanded in vivo and had improved persistence. A single dose (D4) of 2B4.ζ/sIL15 CAR-NKs demonstrated an initial antitumor response, equivalent to that seen following 3 doses (D4-7-10) of 2B4.ζ CAR-NKs. However, mice treated with IL15-secreting NKs had short survival (Fig. 2). Compared to control mice, peripheral blood analysis showed increasing systemic hIL15 and higher levels of hTNFα. In our more aggressive MOLM-13 xenograft model, both single dose 2B4.ζ/sIL15 CAR-NK and multiple dose 2B4.ζ CAR-NK treatment prolonged survival compared to treatment with unmodified NKs. (27 and 26 vs 20 days; n=5 mice; p<0.01; Fig. 2). Conclusion: 2B4.ζ CAR-NKs have limited antitumor efficacy and short persistence in vivo. NK cells armored with secreted IL15 have enhanced anti-AML cytotoxicity and in vitro persistence. Introduction of IL15 secretion confers a distinctly activated phenotype that is maintained during chronic antigen stimulation. Constitutive local IL15 secretion improves in vivo NK cell persistence but may cause lethal toxicity when employed against AML. These results warrant further study and should impact the development of CAR-NK clinical products for patients with AML. Figure 1 Figure 1. Disclosures Ho: Rodeo Therapeutics/Amgen: Patents & Royalties; Exelixis: Consultancy; Sanofi: Research Funding. Bonifant: Kiadis Pharma: Research Funding; BMS: Research Funding; Merck, Sharpe, Dohme: Research Funding.

scholarly journals NF-kB c-Rel is dispensable for the development but is required for the cytotoxic function of NK cells

2021 ◽  
Author(s):  
Y Vicioso ◽  
K Zhang ◽  
Parameswaran Ramakrishnan ◽  
Reshmi Parameswaran
Keyword(s):  
Nk Cells ◽  
Nk Cell ◽  
Granzyme B ◽  
Conditional Knockout ◽  
Cytotoxic Lymphocytes ◽  
And Control ◽  
Cytotoxic Function

AbstractNatural Killer (NK) cells are cytotoxic lymphocytes critical to the innate immune system. We found that germline deficiency of NF-kB c-Rel results in a marked decrease in cytotoxic function of NK cells, both in vitro and in vivo, with no significant differences in the stages of NK cell development. We found that c-Rel binds to the promoters of perforin and granzyme B, two key proteins required for NK cytotoxicity, and controls their transactivation. We generated a NK cell specific c-Rel conditional knockout to study NK cell intrinsic role of c-Rel and found that both global and conditional c-Rel deficiency leads to decreased perforin and granzyme B expression and thereby cytotoxic function. We also confirmed the role of c-Rel in perforin and granzyme B expression in human NK cells. c-Rel reconstitution rescued perforin and granzyme B expressions in c-Rel deficient NK cells and restored their cytotoxic function. Our results show a previously unknown role of c-Rel in transcriptional regulation of perforin and granzyme B expressions and control of NK cell cytotoxic function.

scholarly journals CD33 Delineates Two Functionally Distinct NK Cell Populations Divergent in Cytokine Production and Antibody-Mediated Cellular Cytotoxicity

2022 ◽  
Vol 12 ◽  
Author(s):  
Maryam Hejazi ◽  
Congcong Zhang ◽  
Sabrina B. Bennstein ◽  
Vera Balz ◽  
Sarah B. Reusing ◽  
...  
Keyword(s):  
Nk Cells ◽  
Cytokine Production ◽  
Nk Cell ◽  
Functional Divergence ◽  
Target Cells ◽  
Cellular Cytotoxicity ◽  
Transcriptional Signature ◽  
Promising Target

The generation and expansion of functionally competent NK cells in vitro is of great interest for their application in immunotherapy of cancer. Since CD33 constitutes a promising target for immunotherapy of myeloid malignancies, NK cells expressing a CD33-specific chimeric antigen receptor (CAR) were generated. Unexpectedly, we noted that CD33-CAR NK cells could not be efficiently expanded in vitro due to a fratricide-like process in which CD33-CAR NK cells killed other CD33-CAR NK cells that had upregulated CD33 in culture. This upregulation was dependent on the stimulation protocol and encompassed up to 50% of NK cells including CD56dim NK cells that do generally not express CD33 in vivo. RNAseq analysis revealed that upregulation of CD33+ NK cells was accompanied by a unique transcriptional signature combining features of canonical CD56bright (CD117high, CD16low) and CD56dim NK cells (high expression of granzyme B and perforin). CD33+ NK cells exhibited significantly higher mobilization of cytotoxic granula and comparable levels of cytotoxicity against different leukemic target cells compared to the CD33− subset. Moreover, CD33+ NK cells showed superior production of IFNγ and TNFα, whereas CD33− NK cells exerted increased antibody-dependent cellular cytotoxicity (ADCC). In summary, the study delineates a novel functional divergence between NK cell subsets upon in vitro stimulation that is marked by CD33 expression. By choosing suitable stimulation protocols, it is possible to preferentially generate CD33+ NK cells combining efficient target cell killing and cytokine production, or alternatively CD33− NK cells, which produce less cytokines but are more efficient in antibody-dependent applications.

scholarly journals Natural killer (NK) cell-derived extracellular-vesicle shuttled microRNAs control T cell responses

2022 ◽  
Author(s):  
Sara G Dosil ◽  
Sheila Lopez-Cobo ◽  
Ana Rodriguez-Galan ◽  
Irene Fernandez-Delgado ◽  
Marta Ramirez-Huesca ◽  
...  
Keyword(s):  
T Cell ◽  
Nk Cells ◽  
Natural Killer ◽  
Nk Cell ◽  
Extracellular Vesicle ◽  
Target Cells ◽  
Cell Functions ◽  
Target Molecules ◽  
Th1 Polarization

Natural killer (NK) cells recognise and kill target cells undergoing different types of stress. NK cells are also capable of modulating immune responses. In particular, they regulate T cell functions. Small RNA next-generation sequencing of resting and activated NK cells and their secreted EVs led to the identification of a specific repertoire of NK-EV-associated microRNAs and their post-transcriptional modifications signature. Several microRNAs of NK-EVs, namely miR-10b-5p, miR-92a-3p and miR-155-5p, specifically target molecules involved in Th1 responses. NK-EVs promote the downregulation of GATA-3 mRNA in CD4+ T cells and subsequent T-bet de-repression that leads to Th1 polarization and IFN-γ and IL-2 production. NK-EVs also have an effect on monocyte and moDCs function, driving their activation and increased presentation and co-stimulatory functions. Nanoparticle-delivered NK-EV microRNAs partially recapitulate NK-EV effects in vivo. Our results provide new insights on the immunomodulatory roles of NK-EVs that may help to improve their use as immunotherapeutic tools.

Extracellular granzyme A, complexed to proteoglycans, is protected against inactivation by protease inhibitors

Blood ◽  
2000 ◽  
Vol 95 (4) ◽  
pp. 1465-1472 ◽  
Author(s):  
Elisabeth H. A. Spaeny-Dekking ◽  
Angela M. Kamp ◽  
Christopher J. Froelich ◽  
C. Erik Hack
Keyword(s):  
Nk Cells ◽  
Protease Inhibitors ◽  
Antithrombin Iii ◽  
Nk Cell ◽  
Cell Interaction ◽  
Enzyme Linked Immunosorbent Assay ◽  
Target Cells ◽  
Granzyme A ◽  
Cytotoxic Proteins

Granzyme A (GrA) and B (GrB) together with perforin are the main constituents of cytotoxic granules of cytotoxic T lymphocytes (CTLs) and natural killer (NK) cells. The cytotoxic proteins are released to deliver a lethal hit during contact between the CTL or NK cell and target cell. With the use of an enzyme-linked immunosorbent assay for antigenic levels, we showed in a recent study that plasma of patients with activated CTLs and NK cells contain elevated levels of extracellular GrA. In this study, we determined the form and proteolytic capacity of this extracellular GrA detected in plasma. With the use of various assays, we show that part of the extracellular GrA circulates in the mature conformation and is bound to proteoglycans that protect it against inactivation by protease inhibitors, such as antithrombin III and -2-macroglobulin, whereas another part of GrA circulates as a complex with antithrombin III. Finally, with the use of a novel assay for active GrA, we demonstrate that some plasma samples with high levels of extracellular GrA contain active GrA. These results suggest that various forms of extracellular GrA occur in vivo and that the regulation of GrA activity may be modified by proteoglycans. These data support the notion that granzymes may exert extracellular functions distant from the site of CTL or NK cell interaction with their target cells.

The FLT3-Inhibitors Midostaurin, Sunitinib, Sorafenib, and TKI258 Differentially Affect NK Cell-Mediated Immunesurveillance of Acute Myeloid Leukemia.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 3785-3785
Author(s):  
Julia Salih ◽  
Lothar Kanz ◽  
Helmut R Salih ◽  
Matthias Krusch
Keyword(s):  
Nk Cells ◽  
Myeloid Leukemia ◽  
Nk Cell ◽  
Immune Surveillance ◽  
Leukemia Cells ◽  
Target Cells ◽  
Cell Reactivity ◽  
Flt3 Inhibitors ◽  
Ifn Γ

Abstract Abstract 3785 Poster Board III-721 FLT3 is a receptor tyrosine kinase with an important role in hematopoietic progenitor cell survival and proliferation. The discovery of internal tandem duplication mutations (ITD) in FLT3 was a major breakthrough in understanding the role of abnormally activated FLT3 in myeloid transformation. Between 15% and 34% of AML patients show FLT3-ITD mutations, and thus the inhibition of FLT3 in combination with chemotherapeutic agents may be a promising stragety in the treatment of Acute Myeloid Leukemia (AML). Several protein kinase inhibitors (PKI) targeting FLT3 like e.g. Midostaurin, Sunitinib, Sorafenib, and TKI258 are currently under preclinical and/or clinical evaluation (http://clinicaltrials.gov/ct2/results?term=AML+and+FLT3). Since those PKI, besides targeting their eponymous enzyme FLT3, also inhibit signaling via other molecules they may impair the effector function of various components of anti-tumor immunity. NK cells as part of the innate immune system play an important role in the immune surveillance of tumors due to their ability to directly kill target cells and to shape adaptive immune responses by secreting cytokines like IFN-γ. Clinical evidence for the particularly important role of NK cells in leukemia has recently been provided by studies of haploidentical stem cell transplantation (Ruggeri et al., Science 2002). We report here that CD107a expression as a surrogate marker for degranulation of NK cells within PBMC is inhibited by pharmacological concentrations of Sorafenib (10μg/ml) and Midostaurin (2μg/ml), but not by Sunitinib (200ng/ml) and TKI258 (125ng/ml). In line, pharmacological concentrations of Sunitinib and TKI258 did not affect NK cell cytotoxicity and IFN-γ production in cocultures with leukemia cells. Sorafenib and Midostaurin caused a clear concentration-dependent inhibition of NK cell cytokine production in response to target cells both in resting and in IL-2 activated state (92% and 66%, respectively at plasma peak levels). Furthermore, pharmacological concentrations of Sorafenib and Midostaurin also reduced lysis of leukemia cells by NK cells (54% and 58%, respectively, E:T ratio 10:1) and thus generally compromised NK cell reactivity. Analysis of NK cell signaling revealed that Sorafenib, but not Midostaurin decreased phosphorylation of PI3K and ERK which are important regulators of NK cell reactivity. Thus, Midostaurin inhibits yet undefined signaling events which are crucial for NK effector functions, but are independent of the “classical” PI3K – Rac – PAK – MEK – ERK pathway and are presently under study. Moreover, in light of the important role of NK cells in the immune surveillance of leukemia and the differential influence of clinically used FLT3-inhibitors on NK cell functions our data indicate that the choice and dosing of the most suitable compound in the treatment of AML requires further characterization and careful consideration. Disclosures: No relevant conflicts of interest to declare.

Sign in / Sign up

Export Citation Format

Share Document