TIGIT Expression Positively Associates with NK Cell Function in AML Patients

Abstract Natural killer (NK) cells are essential innate immune effectors with promising anti-leukemia activity in acute myeloid leukemia (AML). However, clinical success of applying NK cells in AML treatment has not been achieved. A better understanding of the regulatory mechanisms for NK cell function is important to optimize this therapeutic strategy. T cell immunoglobulin and ITIM domain (TIGIT) is a recently identified inhibitory receptor expressed on T cells and NK cells. Multiple studies including ours have demonstrated its suppressive effect in anti-tumor CD8 T cell response. However whether and how TIGIT impacts NK cells in AML is unknown. Here we performed phenotypic and functional studies on NK cells derived from patients with newly diagnosed AML (n=30). Cells collected from healthy individuals (n=18) were used as controls. TIGIT expression and their contributions to NK cell function in AML were assessed. Peripheral blood samples were first examined by flow cytometry for the frequency of NK cells (defined as CD56+CD3-). The percentage of NK cells among peripheral blood mononuclear cells (PBMCs) in AML patients is comparable with that of healthy controls. In contrast, when we performed functional analysis to assess NK cells for cytokine release upon in vitro stimulation with a human leukemia cell line K562, we observed significantly lower intracellular production of IFN-γ in cells from AML patients compared with that of healthy controls. Consistently NK cells from AML patients expressed less Perforin, indicating a compromised killing capacity. We next evaluated the expression of TIGIT on CD56+CD3- NK cells. As some AML blasts and monocytes also express CD56, we performed multichannel flow cytometry and carefully gated out other cell components when assessing TIGIT expression. To our surprise, we observed a significantly lower frequency of TIGIT-expressing NK cells in AML compared with that of healthy controls (36.82 ±4.543% vs. 48.9±3.818%, P=0.0463). This data indicated that low-TIGIT expression associates with impaired NK cell function and AML progression. We further examined the phenotype and functional status of TIGIT+ NK cells. Expression of activating receptors (CD16 and CD160) and inhibiting receptors (KIR and NKG2A) on TIGIT+ vs. TIGIT- NK cells were analyzed. We observed a significant higher expression of CD16 (51.27±9.009% vs. 20.63±5.334%, P=0.0001) and CD160 (39.84±6.447% vs. 21.24±4.287%, P=0.0103) on TIGIT+ NK cells compared with that of TIGIT- NK cells. By contrast, TIGIT+ NK cells expressed lower KIR (24.06±3.796% vs. 43.59±6.96%, P=0.0046) and NKG2A (7.658±1.717% vs. 18.68±4.256%, P=0.0167) than TIGIT- NK cells. Importantly, functional studies demonstrated an elevated expression of Granzyme B and increased cytokine (IFN-γ and TNF-α) production by TIGIT+ NK cells compared with TIGIT- NK cells (IFN-γ, P=0.0283; TNF-α P=0.0347; Granzyme B, P=0.0493). These data suggest that TIGIT expression on NK cells associated with activated and high functional status. Collectively, our study demonstrates that 1) in line with lower capacity to produce IFN-γ, NK cells from AML patients express less frequency of TIGIT compared with healthy individuals; 2) TIGIT+ NK cells from AML patients express high levels of activating receptors and are highly functional manifested by more cytokine production and enhanced expression of Granzyme B compared with TIGIT- NK cells. These results indicate that in AML patient, TIGIT may contribute to the upregulation of NK cell function. This is in contrast to the observations of CD8 T cells in which TIGIT plays a suppressive role. Targeting TIGIT for cancer treatment is currently under active development. Our findings bring a call for caution on the TIGIT-targeted therapeutic strategy in AML as TIGIT might be a double-edged sword in anti-leukemia immune regulation. Disclosures No relevant conflicts of interest to declare.

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Effect of interleukins (IL-2, IL-15, IL-18) on receptors activation and cytotoxic activity of natural killer cells in breast cancer cell

African Health Sciences ◽

10.4314/ahs.v20i2.36 ◽

2020 ◽

Vol 20 (2) ◽

pp. 822-832 ◽

Cited By ~ 2

Author(s):

Wahyu Widowati ◽

Diana K Jasaputra ◽

Sutiman B Sumitro ◽

Mochammad A Widodo ◽

Tjandrawati Mozef ◽

...

Keyword(s):

Breast Cancer ◽

Cytotoxic Activity ◽

Nk Cells ◽

Natural Killer ◽

Nk Cell ◽

Cell Receptor ◽

Mcf7 Cells ◽

Activating Receptors ◽

Tnf Α ◽

Ifn Γ

Introduction: Breast cancer is one of the leading cause of cancer deaths in women. Metastasis in BC is caused by immuno- surveillance deficiency, such NK cell maturation, low NK activity and decreasing cytotoxicity. This study was performed to improve activating receptors and cytotoxicity of NK cells using interleukins (ILs). Methods: Human recombinant IL-2, -15, and -18 were used to induce NK cells. We measured the activating and inhibiting receptors, proliferation activity of NK cells, and the cytotoxicity of NK cells on BC cells (MCF7). The effects of ILs were tested on the NK cell receptors CD314, CD158a and CD107a with flowcytometry, proliferation at various incubation times with 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxy methoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assay and concen- trations of TNF-α and IFN-γ by NK cells with ELISA. Results: ILs increased NK cell receptor levels (CD314, CD158a, and CD107a) at 24 hours of incubation. ILs increased NK cell viability, which increased with longer incubation. Moreover, ILs-induced NK cells inhibited proliferation in MCF7 cells, as well as increased TNF-α, IFN-γ, PRF1 and GzmB secretion. Conclusion: IL-2, IL-15, and IL-18 improved activating receptors and proliferation of NK cells. IL-induced NK cells in- creased TNF-α, IFN-γ, PRF1 and GzmB secretion and cytotoxic activity on BC cells. High NK cell numbers increased BC cell growth inhibition. Keywords: Activator; breast cancer; interleukins; natural killer; receptor.

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Echinococcus multilocularis induces surface high expression of inhibitory killer immunoglobulin‐like receptor on natural killer cells

Allergologia et Immunopathologia ◽

10.15586/aei.v49i5.465 ◽

2021 ◽

Vol 49 (5) ◽

pp. 78-86

Author(s):

Bayindala ◽

He Huang ◽

Song Gao ◽

Xinjian Xu

Keyword(s):

Cell Surface ◽

Nk Cells ◽

Natural Killer ◽

Echinococcus Multilocularis ◽

Nk Cell ◽

Single Cells ◽

Granzyme B ◽

Inhibitory Receptor ◽

Tnf Α ◽

Ifn Γ

Alveolar echinococcosis (AE) is a malignant and fatal parasitic disease caused by the larvae of Echinococcus multilocularis (E. multilocularis), which inhibits the activity and proliferation of natural killer (NK) cells. In this study, the functional alteration of hepatic NK cells and their related molecules were studied. The AE-infected patient’s tissue was fixed with formalin, embedded in paraffin, and stained with Masson’s trichrome or hematoxylin and eosin (H&E). Single cells from AE-infected patient or E. multilocularis-infected mice were blocked with Fc-receptor (FcR), and stained with monoclonal antibodies, including CD16, CD56, CD3, KIR2DL1, granzyme B, perforin, Interferon gamma (IFN-γ), and tumor necrosis factor-α (TNF- α) or isotype control, to measure molecules and cytokines of NK cells and analyzed by flow cytometry. The Sirius red staining was used to quantitate hepatic fibrosis by calculating quantitative collagen deposition. AE can adjust both the number of hepatic CD56+ NK cells andits KIR2DL1 expression processes. Moreover, the overexpression of KIR2DL1 in NK cells could downregulate the functioning of immune cells in the liver area close to parasitic lesions. The number and dysfunction of NK cells in E. multilocularis infection could be related to the molecule dynamics of cell surface inhibitory receptor Ly49A, leading to hepatic damage and progression of fibrosis. This study illustrated significant increase in hepatic fibrogenesisand apparent upregulation of hepatic CD56+ NK cell population and its KIR2DL1 expression in AE-infected patients. This opposite variation might be related to the impaired NK cells functioning, such as granzyme B, IFN-γ, and TNF-α secretion. In addition, the cell surface inhibitory receptor Ly49A was related to the intracellular cytokine secretion functions of NK cells.

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Molecular analysis of the methylprednisolone-mediated inhibition of NK-cell function: evidence for different susceptibility of IL-2– versus IL-15–activated NK cells

Blood ◽

10.1182/blood-2006-07-037846 ◽

2007 ◽

Vol 109 (9) ◽

pp. 3767-3775 ◽

Cited By ~ 56

Author(s):

Laura Chiossone ◽

Chiara Vitale ◽

Francesca Cottalasso ◽

Sara Moretti ◽

Bruno Azzarone ◽

...

Keyword(s):

Nk Cells ◽

Cell Function ◽

Nk Cell ◽

Surface Expression ◽

Steroid Treatment ◽

Cross Linking ◽

Target Cells ◽

Activating Receptors ◽

And Function ◽

Nk Cytotoxicity

Abstract Steroids have been shown to inhibit the function of fresh or IL-2–activated natural killer (NK) cells. Since IL-15 plays a key role in NK-cell development and function, we comparatively analyzed the effects of methylprednisolone on IL-2– or IL-15–cultured NK cells. Methylprednisolone inhibited the surface expression of the major activating receptors NKp30 and NKp44 in both conditions, whereas NK-cell proliferation and survival were sharply impaired only in IL-2–cultured NK cells. Accordingly, methylprednisolone inhibited Tyr phosphorylation of STAT1, STAT3, and STAT5 in IL-2–cultured NK cells but only marginally in IL-15–cultured NK cells, whereas JAK3 was inhibited under both conditions. Also, the NK cytotoxicity was similarly impaired in IL-2– or IL-15–cultured NK cells. This effect strictly correlated with the inhibition of ERK1/2 Tyr phosphorylation, perforin release, and cytotoxicity in a redirected killing assay against the FcRγ+ P815 target cells upon cross-linking of NKp46, NKG2D, or 2B4 receptors. In contrast, in the case of CD16, inhibition of ERK1/2 Tyr phosphorylation, perforin release, and cytotoxicity were not impaired. Our study suggests a different ability of IL-15–cultured NK cells to survive to steroid treatment, thus offering interesting clues for a correct NK-cell cytokine conditioning in adoptive immunotherapy.

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Contribution of Natural Killer Cells to Inhibition of Angiogenesis by Interleukin-12

Blood ◽

10.1182/blood.v93.5.1612 ◽

1999 ◽

Vol 93 (5) ◽

pp. 1612-1621 ◽

Cited By ~ 127

Author(s):

Lei Yao ◽

Cecilia Sgadari ◽

Keizo Furuke ◽

Eda T. Bloom ◽

Julie Teruya-Feldstein ◽

...

Keyword(s):

Endothelial Cells ◽

Nk Cells ◽

Natural Killer ◽

Cell Function ◽

Nk Cell ◽

Primary Cultures ◽

Interleukin 12 ◽

Ifn Γ

Abstract Interleukin-12 (IL-12) inhibits angiogenesis in vivo by inducing interferon-γ (IFN-γ) and other downstream mediators. Here, we report that neutralization of natural killer (NK) cell function with antibodies to either asialo GM1 or NK 1.1 reversed IL-12 inhibition of basic fibroblast growth factor (bFGF)-induced angiogenesis in athymic mice. By immunohistochemistry, those sites where bFGF-induced neovascularization was inhibited by IL-12 displayed accumulation of NK cells and the presence of IP-10–positive cells. Based on expression of the cytolytic mediators perforin and granzyme B, the NK cells were locally activated. Experimental Burkitt lymphomas treated locally with IL-12 displayed tumor tissue necrosis, vascular damage, and NK-cell infiltration surrounding small vessels. After activation in vitro with IL-12, NK cells from nude mice became strongly cytotoxic for primary cultures of syngeneic aortic endothelial cells. Cytotoxicity was neutralized by antibodies to IFN-γ. These results document that NK cells are required mediators of angiogenesis inhibition by IL-12, and provide evidence that NK-cell cytotoxicity of endothelial cells is a potential mechanism by which IL-12 can suppress neovascularization.

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

Blood ◽

10.1182/blood-2005-04-1644 ◽

2005 ◽

Vol 106 (13) ◽

pp. 4370-4376 ◽

Cited By ~ 150

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.

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Tim-3, a Novel Immune Receptor, Is Constitutively Expressed on Human Natural Killer Cells and Functions as An Activating Coreceptor

Blood ◽

10.1182/blood.v116.21.106.106 ◽

2010 ◽

Vol 116 (21) ◽

pp. 106-106

Author(s):

Michelle Gleason ◽

Todd Lenvik ◽

Valarie McCullar ◽

Sarah Cooley ◽

Michael Verneris ◽

...

Keyword(s):

Nk Cells ◽

Board Of Directors ◽

Cell Activation ◽

Low Dose ◽

Cell Function ◽

Nk Cell ◽

Effector Function ◽

Advisory Committees ◽

Immune Receptor ◽

Ifn Γ

Abstract Abstract 106 NK cells are an attractive option for immunotherapy as they do not require pre-sensitization for anti-tumor activity and do not induce graft versus host disease (GvHD) in an allogeneic transplant setting. The potential of NK cells in controlling human hematological malignancies has been increasingly recognized in recent years, as the adoptive transfer of alloreactive NK cells in hematopoietic cell transplantation (HCT) clinical trials have demonstrated therapeutic anti-leukemia effects. NK cell function is regulated by the integration of antagonist signals received from cell surface activating and inhibitory receptors. Tim-3 is a novel immune receptor that is a member of the T cell immunoglobulin and mucin-containing domain (TIM) family of glycoproteins. While its role in T cells and antigen presenting cells has been described, little is known about its function in human NK cells. While Tim-3 is present on a variety of immune cells, resting NK cells constitutively express Tim-3 compared to other lymphocyte populations (NK: 73±3%; NKT: 6±1%; T: 1±1%; n=14) and we hypothesized that Tim-3 may be important in mediating NK cell function. The unique subset of cytokine producing CD56Bright NK cells exhibited significantly lower resting Tim-3 expression compared to CD56Dim NK cells (53±3% vs. 75±3%; p<0.001, n=14). Distinct Tim-3 expression patterns were found on resting CD56Dim NK cells and activation with low dose IL-12 (1ng/mL) and IL-18 (10ng/mL), intended to more closely mimic physiologic conditions, resulted in further differentiation of this unique expression pattern dividing NK cells into 4 distinct populations: Tim-3 was homogeneously up-regulated on all CD56Bright NK cells after activation while CD56Dim NK cells were further stratified into 3 defined populations with Tim-3hi, Tim-3lo and Tim-3neg expression. The only identified ligand of Tim-3 is galectin-9 (Gal-9), a β-galactoside binding lectin, which is expressed on a wide range of healthy and malignant cells. To investigate the potential function of Tim-3, an expression vector containing human Gal-9 was transduced into K562 and Raji cells, both without endogenous Gal-9 expression. Resting NK cytotoxicity (51Cr release) was found to be increased in the presence of Gal-9 compared to the non-Gal-9 expressing targets [E:T=0.7:1, K562 vs. K562-Gal-9: 25±3% vs. 33±3% (n=8, p<0.05); E:T=20:1, Raji vs. Raji-Gal-9: 8±1% vs. 17±2% (n=4, p<0.05)]. Analysis of CD107a degranulation showed that resting Tim-3+ CD56Bright cells were more functional against Gal-9 expressing targets than Tim-3− CD56Bright cells, suggesting that Tim-3 might also play a role in IFN-γ production. To further investigate this, resting NK cells were activated with low-dose IL-12/IL-18 overnight and IFN-γ levels were measured in response to soluble rhGal-9 (0, 2.5, 5, 10 and 20nM). Exposure to soluble rhGal-9 alone without IL-12/IL-18 did not induce IFN-γ production. For both the CD56Bright and CD56Dim IL-12/IL-18 activated NK populations, only Tim-3+ NK cells displayed a dose dependent increase in IFN-γ production upon exposure to soluble rhGal-9 compared to Tim-3− NK cells. To understand the relevance of the distinct Tim-3 populations circulating in resting blood, CD56Bright, CD56Dim/Tim-3hi, CD56Dim/Tim-3lo and CD56Dim/Tim-3neg populations were sorted, cultured overnight in IL-12/IL-18 and exposed to soluble rhGal-9. Results showed the Tim-3 expressing populations contain the predominant IFN-γ producing cells that were responsive to rhGal-9 (results for the sorted CD56Dim/Tim-3lo population shown in the figure below). This increase in IFN-γ production within the Tim-3 expressing NK cell populations was abrogated by the addition of β-lactose, a β-galactoside that binds and blocks Gal-9 activity. Lastly, Western blot and immunohistochemistry analysis of human primary acute leukemia blasts revealed high Gal-9 expression. As the presence of ligands for NK cell activating receptors on tumors provide an important prerequisite for NK cell activation and effector function, we show a novel functional role for the receptor Tim-3 in human NK cell biology in the presence of its ligand Gal-9. We, therefore, propose a model where constitutively expressed Tim-3 is up-regulated by NK cell activation and effector function is enhanced by Tim-3/Gal-9 interaction, which may potentiate the elimination of Gal-9 positive tumors by NK cells. Disclosures: Niki: GalPharma: Membership on an entity's Board of Directors or advisory committees. Hirashima:GalPharma: Membership on an entity's Board of Directors or advisory committees.

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The JAK1/JAK2 Inhibitor Ruxolitinib Substantially Affects NK Cell Biology

Blood ◽

10.1182/blood.v122.21.16.16 ◽

2013 ◽

Vol 122 (21) ◽

pp. 16-16 ◽

Cited By ~ 5

Author(s):

Kathrin Schönberg ◽

Janna Rudolph ◽

Isabelle Cornez ◽

Peter Brossart ◽

Dominik Wolf

Keyword(s):

Flow Cytometry ◽

Cell Proliferation ◽

Nk Cells ◽

Cell Activation ◽

Nk Cell ◽

Granzyme B ◽

Receptor Activation ◽

Jak2 Inhibitor ◽

Signaling Events ◽

Ifn Γ

Abstract Introduction We recently demonstrated that ruxolitinib (INCB018424), the first approved JAK1/JAK2 inhibitor for treatment of myelofibrosis (MF), exerts potent anti-inflammatory activity. This may at least in part explain higher infection rates observed in ruxolitinib-treated patients. NK cells are critical for cancer-immune surveillance and cytokine-mediated signals are central for proper NK cell activation. We here aimed to characterize in detail the effects of JAK1/2 inhibition on human NK cells. Methods Highly purified CD56+ NK cells were isolated from human peripheral buffy coats by magnetic bead isolation and subsequently exposed to increasing concentrations of ruxolitinib (0.1-10 µM). Cytokine (1000U/ml IL-2, 25ng/ml IL-15)-induced NK cell proliferation was analyzed by CFSE dilution. Phenotypic and functional NK cell activation markers (NKp46, NKG2D, Granzyme B, CD16, and CD69) were analyzed by flow cytometry (including CD107a expression for degranulation). NK cell function was tested by flow-cytometry-based killing assays and quantification of IFN-γ production upon stimulation with either MHC class I-deficient K562 target cells or cytokines (IL-12, IL-18). In addition, phenotypic and functional analyses were also tested during NK receptor activation via plate-bound activating NKp46 antibodies. Signaling events were analyzed by Western Blot analysis to detect phosphorylation of JAK1 and JAK2 as well as by applying phospho-flow technology to evaluate ruxolitinib-mediated changes of cytokine-dependent signalling cascades (pS6, pSTAT1, pSTAT3, pSTAT5, pERK, pAKT, pP38, and pZAP70). Results Our results demonstrate provide first evidence that ruxolitinib profoundly affects cytokine-induced NK cell activation. This includes a significant and dose-dependent reduction of NK cell proliferation, reduced induction of activation-associated surface markers (including NKp46, NKG2D, Granzyme B, CD16, CD69) as well as impaired killing activity against the classical NK target cell line K562. In addition, all main functional activities of NK cells are down-regulated as shown by reduced cytotoxic capacity, impaired degranulation and IFN-γ production. After wash-out, the inhibitory effects of ruxolitinib on NK cells are fully reversible, as shown by proper re-activation by cytokines. In contrast to cytokine-mediated NK cell activation, stimulation via the NK-specific receptor NKp46 are not affected by ruxolitinib. Of note, ruxolitinib does not affect NK cell viability. On a molecular level, phospho-flow analyses revealed that cytokine associated signaling events, such as phosphorylation of STAT5 and S6 were dose-dependently reduced by ruxolitinib in primary human NK cells. Conclusions Ruxolitinib strongly inhibits NK cell activation leading to impaired proliferation and functional activity. Experiments verifying these effects in patients are currently ongoing and will be presented at the meeting. Our findings may have important clinical implications, when considering the application of ruxolitinib as GvHD therapy, because NK cells are critically involved in the GvL effect after allogeneic stem cell transplantation. Disclosures: Wolf: Novartis: Honoraria, Research Funding.

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Early Disease Relapse after Tyrosine Kinase Inhibitor Treatment Discontinuation in CML Is Related Both to Low Number and Impaired Function of NK-Cells

Blood ◽

10.1182/blood.v124.21.812.812 ◽

2014 ◽

Vol 124 (21) ◽

pp. 812-812 ◽

Cited By ~ 7

Author(s):

Mette Matilda Ilander ◽

Ulla Olsson-Strömberg ◽

Hanna Lähteenmäki ◽

Kasanen Tiina ◽

Perttu Koskenvesa ◽

...

Keyword(s):

T Cells ◽

Nk Cells ◽

Research Funding ◽

Nk Cell ◽

Treatment Discontinuation ◽

Disease Relapse ◽

Cell Counts ◽

Tnf Α ◽

Ifn Γ

Abstract Background: Recent reports suggest that approximately 40% of CML patients who have achieved sustained complete molecular remission are able to stop TKI treatment without disease relapse. However, there are no predictive markers for successful therapy discontinuation. Therefore, we set up an immunological sub-study in the ongoing pan-European EURO-SKI stopping study. Our aim was to identify predictive biomarkers for relapse/non-relapse and to understand more on the mechanisms of immune surveillance in CML. Methods: The EURO-SKI study started in 2012, and patients included were at least three years on TKI and at least one year in MR4 or deeper before the study entry. Basic lymphocyte immunophenotyping (the number of NK-, T- and B-cells) was performed at the time of therapy discontinuation and 1, 6, and 12 months after the TKI stop and in case of relapse (defined as loss of MMR, BCR-ABL1>0.1% IS). In addition, from a proportion of patients more detailed immunophenotypic and functional analyses (cytotoxicity of NK-cells and secretion of Th1 type of cytokines IFN-γ/TNF-α) were done at the same times. Results: Thus far 119 Nordic patients (imatinib n=105, dasatinib n=12, nilotinib n=2) who have discontinued TKI treatment within the EURO-SKI study have been included in the lymphocyte subclass analysis (results are presented from patients who have reached 6 months follow-up). Immunophenotyping analysis demonstrates that imatinib treated patients who were able to maintain remission for 6 months (n=36) had increased NK-cell counts (0.26 vs. 0.15x109cells/L, p=0.01, NK-cell proportion 18.9% vs. 11%, p=0.005) at the time of drug discontinuation compared to patients who relapsed early (before 5 months n=22). Furthermore, the phenotype of NK-cells was more cytotoxic (more CD57+ and CD16+cells and less CD62L+cells), and also their IFN-γ/TNF-α secretion was enhanced (19.2% vs. 13%, p=0.02). Surprisingly, patients who relapsed more slowly (after 5 months, n=16) had similar baseline NK-cell counts (0.37x109cells/L), NK-cell proportion (21.2%), and phenotype and function as patients, who were able to stay in remission. No differences in the NK-cell counts were observed between patients who had detectable or undetectable BCR-ABL1 transcripts at the baseline (0.22 x109cells/L vs. 0.31 x109cells/L, p=0.61). Interestingly, NK-cell count was higher in patients with low Sokal risk score than in patients with intermediate risk (0.33 x109cells/L vs. 0.20 x109cells/L, p=0.04). Furthermore, there was a trend that male patients had a higher proportion of NK-cells than females (21.6% vs. 15.7%, p=0.06). Pretreatment with IFN-α or the duration of imatinib treatment did not have an effect on NK-cell count or proportion. In comparison to the imatinib group, dasatinib treated patients had higher NK-cell counts at the baseline (median 0.52x109cells/L vs. 0.26x109cells/L, p=0.02), and also the proportion of CD27 (median 50% vs. 16%, p=0.01) and CD57 expressing (median 79% vs. 74%, p=0.05) NK-cells was higher. The follow-up time of dasatinib treated patients is not yet long enough to correlate the NK-cell counts with the success of the treatment discontinuation. The absolute number of T-cells or their function did not differ significantly between relapsing and non-relapsing patients at the time of treatment discontinuation. However, both CD4+ and CD8+ T-cells tended to be more mature in patients who stayed in remission compared to patients who relapsed early (CD4+CD57+CD62L- median 5.7% vs. 2.4%, p=0.06, CD8+CD62L+CD45RA+ 13% vs. 26.7%, p=0.05). The analysis of follow-up samples showed that in patients who stayed in remission the Th1 type cytokine (IFN-γ/TNF-α) secretion of CD8+T-cells increased at 6 months compared to baseline (23.6 vs. 18.5%, p=0.07). Same phenomenon was observed in the late relapsing group at relapse compared to baseline (37.9 vs. 13.5%, p=0.03). No similar increase was observed in the early relapsing group. Conclusions: Low NK-cell numbers and poor cytokine secretion may predict early disease relapse after TKI discontinuation. However, patients who relapse later have high numbers of normally functioning NK-cells. Further research (detailed phenotypic analysis of NK- and T-cells including activating and inhibitory receptors and immune checkpoint molecules) and correlation of biomarker data with clinical parameters are ongoing to understand the ultimate determining factors of relapse. Disclosures Själander: Novartis: Honoraria. Hjorth-Hansen:Novartis: Honoraria; Bristol-myers Squibb: Honoraria; Ariad: Honoraria; Pfizer: Honoraria. Porkka:BMS: Honoraria; BMS: Research Funding; Novartis: Honoraria; Novartis: Research Funding; Pfizer: Research Funding. Mustjoki:Bristol-Myers Squibb: Honoraria, Research Funding; Novartis: Honoraria, Research Funding.

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A family with Papillon-Lefèvre syndrome reveals a requirement for cathepsin C in granzyme B activation and NK cell cytolytic activity

Blood ◽

10.1182/blood-2005-03-1140 ◽

2006 ◽

Vol 107 (9) ◽

pp. 3665-3668 ◽

Cited By ~ 49

Author(s):

Josephine L. Meade ◽

Erika A. de Wynter ◽

Peter Brett ◽

Saghira Malik Sharif ◽

C. Geoffrey Woods ◽

...

Keyword(s):

Nk Cells ◽

Cell Function ◽

Nk Cell ◽

Granzyme B ◽

Interleukin 2 ◽

Target Cells ◽

Loss Of Function ◽

Cathepsin C ◽

Cytolytic Function

Activation of granzyme B, a key cytolytic effector molecule of natural killer (NK) cells, requires removal of an N-terminal pro-domain. In mice, cathepsin C is required for granzyme processing and normal NK cell cytolytic function, whereas in patients with Papillon-Lefèvre syndrome (PLS), loss-of-function mutations in cathepsin C do not affect lymphokine activated killer (LAK) cell function. Here we demonstrate that resting PLS NK cells do have a cytolytic defect and fail to induce the caspase cascade in target cells. NK cells from these patients contain inactive granzyme B, indicating that cathepsin C is required for granzyme B activation in unstimulated human NK cells. However, in vitro activation of PLS NK cells with interleukin-2 restores cytolytic function and granzyme B activity by a cathepsin C-independent mechanism. This is the first documented example of a human mutation affecting granzyme B activity and highlights the importance of cathepsin C in human NK cell function.

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Combined Stimulation with Interleukin-18 and Interleukin-12 Potently Induces Interleukin-8 Production by Natural Killer Cells

Journal of Innate Immunity ◽

10.1159/000477172 ◽

2017 ◽

Vol 9 (5) ◽

pp. 511-525 ◽

Cited By ~ 5

Author(s):

Sophie M. Poznanski ◽

Amanda J. Lee ◽

Tina Nham ◽

Evan Lusty ◽

Margaret J. Larché ◽

...

Keyword(s):

Immune Response ◽

Nk Cells ◽

Natural Killer ◽

Peripheral Blood ◽

Mononuclear Cells ◽

Nk Cell ◽

Critical Role ◽

Interleukin 12 ◽

Tnf Α ◽

Ifn Γ

The combination of interleukin (IL)-18 and IL-12 (IL-18+IL-12) potently stimulates natural killer (NK) cells, triggering an innate immune response to infections and cancers. Strategies exploiting the effects of IL-18+IL-12 have shown promise for cancer immunotherapy. However, studies have primarily characterized the NK cell response to IL-18+IL-12 in terms of interferon (IFN)-γ production, with little focus on other cytokines produced. IL-8 plays a critical role in activating and recruiting immune cells, but it also has tumor-promoting functions. IL-8 is classically produced by regulatory NK cells; however, cytotoxic NK cells do not typically produce IL-8. In this study, we uncover that stimulation with IL-18+IL-12 induces high levels of IL-8 production by ex vivo expanded and freshly isolated NK cells and NK cells in peripheral blood mononuclear cells. We further report that tumor necrosis factor (TNF)-α, produced by NK cells following IL-18+IL-12 stimulation, regulates IL-8 production. The IL-8 produced is in turn required for maximal IFN-γ and TNF-α production. These findings may have important implications for the immune response to infections and cancer immunotherapies. This study broadens our understanding of NK cell function and IL-18+IL-12 synergy by uncovering an unprecedented ability of IL-18+IL-12-activated peripheral blood NK cells to produce elevated levels of IL-8 and identifying the requirement for intermediates induced by IL-18+IL-12 for maximal cytokine production following stimulation.

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