Accepted Manuscript Regulatory CD4+ T cells (Tregs) Modulate the Interaction between NK Cells and Hepatic Stellate Cells (HSC) by Acting on either Cell Type Bettina Langhans, Abdel Wahed Alwan, Benjamin Krämer, Andreas Glässner, Philipp Lutz, Christian P. Strassburg, Jacob Nattermann, Ulrich Spengler PII: DOI: Reference:

S0168-8278(14)00626-6 http://dx.doi.org/10.1016/j.jhep.2014.08.038 JHEPAT 5318

To appear in:

Journal of Hepatology

Received Date: Revised Date: Accepted Date:

14 March 2014 8 July 2014 26 August 2014

Please cite this article as: Langhans, B., Alwan, A.W., Krämer, B., Glässner, A., Lutz, P., Strassburg, C.P., Nattermann, J., Spengler, U., Regulatory CD4+ T cells (Tregs) Modulate the Interaction between NK Cells and Hepatic Stellate Cells (HSC) by Acting on either Cell Type, Journal of Hepatology (2014), doi: http://dx.doi.org/ 10.1016/j.jhep.2014.08.038

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REGULATORY CD4+ T CELLS (TREGS) MODULATE THE INTERACTION BETWEEN NK CELLS AND HEPATIC STELLATE CELLS (HSC) BY ACTING ON EITHER CELL TYPE

Bettina Langhans, Abdel Wahed Alwan, Benjamin Krämer, Andreas Glässner, Philipp Lutz, Christian P. Strassburg, Jacob Nattermann, Ulrich Spengler

Department of Internal Medicine I, University of Bonn, Bonn, Germany and the German Centre of Infection Research (DZIF), partner site Cologne-Bonn, Bonn

Correspondence:

PD Dr. rer. nat. Bettina Langhans Department of Internal Medicine I University of Bonn Sigmund-Freud-Straße 25 53105 Bonn, Germany Tel: ++49-228-287-51416 Fax: ++49-228-287-51419 e-mail: [email protected]

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Electronic word count: 4992 words Number of figures and tables: 4 figures, 1 table List of Abbreviations:

CTLA-4, cytotoxic T-Lymphocyte antigen 4 Foxp3, forkhead box P3 GITR, glucocorticoid-induced TNFR family related gene HCV, hepatitis C virus HLA, human leukocyte antigen HSC, primary human hepatic stellate cells IL-8, interleukin-8 IL-10, interleukin-10 MIC-A/B, MHC class I polypeptide-related sequence A/B NK cells, natural killer cells PD-1, programmed cell death protein 1 TGF-ß1, transforming growth factor beta1 TIM3, T-cell immunoglobulin domain and mucin domain 3 Tregs, CD4+ regulatory T cells

Conflict of interest: The authors do not have a commercial or any other associations that might pose a conflict of interest.

Financial support: This work was supported by the German Centre of Infection Research (DZIF) and the German Research Foundation (DFG; grant SP483/4-1/2 to U.S. and SFB/TRR57 to J. N. and U.S.).

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Abstract Background & Aims: NK cells regulate liver fibrosis by killing activated hepatic stellate cells (HSC) and are controlled themselves by immune cells and/or soluble factors. Here, we analyzed if CD4+ regulatory T cells (Tregs) modify the interaction between NK cells and HSC. Methods: Modification of NK cell activity against HSC was studied in CD56highCD16NK cells using a flow cytometric CD107a degranulation assay and co-cultures with Tregs from healthy donors and patients with hepatitis C, respectively. We studied underlying mechanisms in detail applying Treg-supernatants, Treg-pre-treated HSC, and recombinant IL-8, TGF-ß1 and IL-10 as well as blocking experiments with neutralizing antibodies and analyzed Treg-associated changes in the expression of NK cell receptor ligands on HSC. Results: Tregs suppressed NK cell activation during HSC co-culture in a cellcontact-dependent manner involving CTLA-4. NK cell degranulation was further reduced, when HSC had been pre-treated with Tregs (p=0.043), Treg-supernatants (p=0.001)

or rIL-8

(R=0.630, p=0.001) and rTGF-ß1 (R=0.608,

p=0.002),

respectively. This additional inhibitory effect corresponded to IL-8/TGF-ß1-mediated down-regulation of MIC-A/B and HLA class-I on HSC. Tregs from hepatitis C likewise inhibited NK cell activity, which was reversed significantly in specific blocking experiments. Discussion: Our data indicate that Tregs interfere with NK cell regulation of fibrogenesis via both direct cell-contact-dependent inhibition of NK cells and release of soluble factors down-regulating activating NK cell receptor ligands on HSC. Our data may be particularly relevant for intrahepatic accumulation of Tregs in chronic hepatitis C because down-regulated NK cell activity against HSC may blunt their control of fibrogenesis. 3

Introduction HCV (hepatitis C virus) is a global health problem since most patients with HCV infection fail to eradicate the virus and carry a substantial risk to progress towards liver fibrosis/cirrhosis. The exact mechanisms leading to liver fibrosis are still largely unknown. However, there is accumulating evidence that immune cells pivotally modulate fibrogenesis of hepatitis C [1, 2]. CD4+ regulatory T cells (Treg) and natural killer (NK) cells are key players in the immune system which regulate liver fibrosis [3, 4]. NK cells exert important anti-fibrotic activity on activated hepatic stellate cells (HSC) [5-9]. In the mouse retinoid acid signaling sensitizes HSC to NK cell killing via upregulation of the NKG2D ligand RAE1 [5]. In man HSC activation likewise leads to up-regulation of NKG2D ligands ULBP-1/2 and MIC-A/B, which trigger NK cell killing [9]. Moreover, reduced NK cell activity has also been linked to accelerated progression of liver fibrosis in ethanol-fed mice [10, 11]. Importantly, anti-fibrotic NK cell activity was inversely correlated with liver fibrosis in patients with chronic hepatitis C and has been reported repeatedly to be impaired in chronic hepatitis C owing to either altered expression of NK cell receptors or increased production of TGF-ß1 by HCV-activated HSC [9, 11-15]. NK cell function is also modified by crosstalk with other immune cells, e.g. CD4+ T cells, and by soluble factors [16]. In this context, it has been reported that CD4+ Tregs interact with NK cells and suppress their effector functions [17, 18]. However, at present it is unclear, if such interactions also affect regulation of hepatic fibrosis by NK cells. Recently, we identified and characterized HCV-specific CD4+ Tregs in chronic hepatitis C [19]. We further demonstrated enrichment of such cells in the livers of patients with advanced fibrosis and provided first evidence that these Tregs enhance fibrosis by releasing IL-8 [20]. In addition, HCV-specific Tregs inhibited effector CD4+ 4

T cells via soluble IL-10 and IL-35 [19]. Here, we analyzed in how far CD4+ Tregs modify the interactions between NK cells and HSC.

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Material and Methods Patients Twelve patients with chronic hepatitis C virus (HCV) infection were enrolled into this study. The clinical features are summarized in table 1. All patients were treatmentnaïve and had detectable HCV RNA. As controls we studied 15 healthy blood donors. The reported studies were approved by the Institutional Review Boards of the Bonn University Ethics Committee (decision #067/10). Written informed consent was obtained from the patients and healthy blood donors prior to sample collection.

Reagents Human recombinant proteins IL-2, IL-8, TGF-ß1, IL-10 and OKT-3 (human anti-CD3) were obtained from R&D Systems (Wiesbaden, Germany), anti-CD28 from BD Biosciences (Heidelberg, Germany). RPMI 1640 medium supplemented with 10% (v/v) human AB serum, 200 mg/ml glutamine, 50 µg/ml gentamycin, 100 units/ml penicillin and 100 µg/ml streptomycin (all from PAA Laboratories, Pasching, Austria) was used for cell culture. The following LEAF™ purified blocking antibodies were used: anti-human CTLA-4 (clone L3D10; Biolegend), anti-human TIM3 (clone F382E2; Biolegend), anti-human PD-1 (clone EH12.2H7; Biolegend), anti-human GITR (clone 621; Biolegend), anti-human TGF-β1 (clone 19/D8; Biolegend), anti-human IL-8 (AF-208-NA; R&D Systems), anti-human IL-10 (AF-217-NA; R&D Systems), and control antibodies (mouse IgG1 k isotype control (Biolegend); antibody AB-108-C (R&D Systems)).

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Cell populations and their in vitro stimulation NK cells and CD4+ effector T cells were isolated from peripheral blood mononuclear cells (PBMC) of healthy donors using a “human NK isolation kit” and “CD4+ Pan T cell isolation Kit II” following the manufacturer’s instruction (both Miltenyi Biotec, Bergisch Gladbach, Germany). We also depleted Tregs from purified CD4+ effector T cells using anti-CD25 Microbeads (Miltenyi Biotec). In addition, we isolated CD4+ Tregs from PBMC of patients with chronic hepatitis C and healthy blood donors using the “human CD4+CD25+CD127dim/-Regulatory T Cell Isolation Kit II” (Miltenyi Biotec). Purity of isolated cell populations was >95% as determined by flow cytometric analysis. Cell populations were cryo-preserved until analysis. Tregs, effector T cells and CD25-depleted effector T cells were stimulated with antiCD3/CD28 (0.5 µg/ml/1 µg/ml) in RPMI 1640. After 24 h T cell subsets or their supernatants were used for co-culture experiments with isolated NK cells in medium supplemented with 25 IU/ml IL-2. Furthermore, we included 9 Treg clones generated from patients with chronic hepatitis

C

as

described

previously

[19,

20,

21].

Treg

clones

were

CD25+Foxp3+CD127low, had poor proliferative capacity and produced IL-10 and IL-8, but no IFN-gamma or IL-4.

Primary human hepatic stellate cells (HSC) Primary human HSC were obtained from ScienCell (San Diego, CA, USA) and were cultured for 2-4 passages in “Stellate Cell Medium” (SteCM, ScienCell) supplemented with 2% fetal bovine serum, 5 ml stellate cell growth supplement, 10 U/ml penicillin and 10 mg/ml streptomycin (all ingredients obtained from ScienCell) at 37°C with 5% CO2. Cryopreserved HSC were thawed and cultured in supplemented SteCM for 2

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days and checked for viability by trypan blue exclusion before they were used in the experiments.

Activation of NK cells by co-culture with primary activated HSC Activation of CD56highCD16- NK cells by HSC was analyzed by studying CD107a degranulation in co-cultures with HSC [9, 13-16, 22], which corresponds to NK cell cytotoxicity (see supplementary figure 1). Briefly, isolated NK cells – pre-stimulated with or without Tregs – were incubated with 30,000 HSC at an effector to target (E:T) ratio of 1:1 in the presence of PE-conjugated CD107a mAb (BioLegend, Fell, Germany). The optimum E:T ratio was determined in prior control experiments using CD25highCD4+ KARPAS-299 [23] as control cells of natural CD4+ Tregs. After 1 h GolgiStop (BD Biosciences) was added, and the cells were cultured for additional 3 h. Then, NK cells were stained with the following antibodies: anti-CD3-PerCP, antiCD56-APC, anti-CD16-FITC, anti-CD69-PE-Cy7 (all BioLegend). Viability of NK cells was checked with eFluor 450 (eBioscience, San Diego, CA, USA). Samples were analyzed on a FACSCanto II (BD Biosciences) with FlowJo 7.2.2 software (TreeStar Inc., Ashland, OR, USA).

Surface expression of NK cell receptor ligands on HSC We also studied expression of surface molecules MHC class-I (HLA-ABC, BD Biosciences), MIC-A/B (R&D Systems) and the control antigen HLA-DR (MHC class II, BioLegend) on the HSC before and after pre-treatment with recombinant cytokines (IL-8, TGF-ß1, IL-10). Expression was determined as relative fluorescence intensity (RFI = mean fluorescence intensity (MFI) with respect to isotype controls).

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Statistics Differences between groups were compared by t-test, non-parametric Mann-Whitney test, and Fisher’s exact test as appropriate. Calculations were done by the SPSS (SPSS 15.0, Chicago, Illinois) and the GraphPad Prism 4.0 (GraphPad Prism, San Diego California, USA) software packages. P-values

Regulatory CD4+ T cells modulate the interaction between NK cells and hepatic stellate cells by acting on either cell type.

NK cells regulate liver fibrosis by killing activated hepatic stellate cells (HSCs) and are controlled themselves by immune cells and/or soluble facto...
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