low dose interleukin 2 promotes stat5 phosphorylation treg survival and ctla 4 dependent function in autoimmune liver diseases

Low dose interleukin-2 promotes STAT5 phosphorylation, Treg survival and CTLA-4 dependent function in autoimmune liver diseases Hannah C Jeffery1, Louisa E Jeffery2, Philipp Lutz1, Margaret Corrigan1, 3, Gwilym J Webb1,3, Gideon M Hirschfield1,3, David H Adams1, 3, Ye Htun Oo1, Centre for Liver Research and National Institute of Health Research Liver Biomedical Research Unit, Institute of Immunology and Immunotherapy, University of Birmingham, UK Institute of Metabolism and Systems Research, University of Birmingham, UK Liver Transplant and Hepatobiliary Unit, University Hospital of Birmingham NHS Foundation Trust, Birmingham, UK Short title: Low dose IL-2 promotes regulatory T cell function in autoimmune liver diseases Correspondence to: Dr Ye Htun Oo, MBBS, MRCP, PhD MRC Clinician Scientist & Consultant Hepatologist Centre for Liver Research and NIHR Birmingham Biomedical Research Unit University of Birmingham, Vincent Drive, Edgbaston, Birmingham, B15 2TT, UK Tel: +44 121 415 8700 Fax: +44 121 415 8701 E-mail: y.h.oo@bham.ac.uk KEY WORDS: Autoimmune Liver Disease, Regulatory T cells, Interleukin2, CTLA-4, Bcl-2, STAT5 Treg, regulatory T cell STAT5, Signal transduction and activator of transcription CTLA-4, Cytotoxic T lymphocyte antigen-4 This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process which may lead to differences between this version and the Version of Record Please cite this article as an ‘Accepted Article’, doi: 10.1111/cei.12940 This article is protected by copyright All rights reserved Clinical Experimental Immunology Page 58 of 105 Acknowledgements: We would like to thank Matthew Graeme MacKenzie at the University of Birmingham Technology Hub Services for his help with cell sorting We would also like to thank medical, surgical and anaesthetic staff from UHB NHS Foundation Trust for their help with tissue acquirement and patients for their donations This article is protected by copyright All rights reserved Page 59 of 105 Clinical Experimental Immunology ABSTRACT: CD4+CD25highCD127lowFOXP3+ regulatory T cells (Treg) are essential for the maintenance of peripheral tolerance Impaired Treg function and an imbalance between effector and regulatory T cells contribute to the pathogenesis of autoimmune diseases We recently reported that the hepatic microenvironment is deficient in IL-2, a cytokine essential for Treg survival and function Consequently, few liver-infiltrating Treg demonstrate STAT5 phosphorylation To establish the potential of IL-2 to enhance Treg therapy, we investigated the effects of very low dose Proleukin (VLDP) on the phosphorylation of STAT5 and the subsequent survival and function of Treg and T effector cells from the blood and livers of patients with autoimmune liver diseases VLDP, less than IU/ml, resulted in selective phosphorylation of STAT5 in Treg but not effector T cells or natural killer cells and associated with increased expression of CTLA-4, FOXP3 and CD25 and the antiapoptotic protein Bcl-2 in Treg with the greatest enhancement of regulatory phenotype in the effector memory Treg population VLDP also maintained expression of the liver-homing chemokine receptor CXCR3 VLDP enhanced Treg function in a CTLA-4 dependent manner These findings open new avenues for future VLDP cytokine therapy alone or in combination with clinical grade Treg in autoimmune liver diseases as VLDP could not only enhance regulatory phenotype and functional property but also the survival of intrahepatic Treg This article is protected by copyright All rights reserved Clinical Experimental Immunology Page 60 of 105 INTRODUCTION: Naturally occurring CD4+CD25highCD127lowFOXP3+ regulatory T cells (Treg) constitute 5–10% of peripheral CD4+ T cells and maintain peripheral self-tolerance in rodents and humans(1, 2) Functional impairment or quantitative deficiency of Tregs has been described in autoimmune liver diseases (AILD)(3, 4) including those targeted at bile ducts (primary biliary cholangitis (PBC)(5, 6), or primary sclerosing cholangitis (PSC)) and hepatocytes (autoimmune hepatitis (AIH))(3, 7-9) Current therapies for AILD are non-curative, provide unsatisfactory control of hepatic inflammation and require long-term immunosuppressive medications that carry unfavorable side effects Thus, autologous Treg therapy is an attractive option for the treatment of AILD that could provide long-term immune-regulation without daily medications and global immunosuppression Treg survival and function is dependent on interleukin-2 (IL-2)(10) which is required for the maintenance of effective levels of functional Treg in autoimmune diseases(11-13) The importance of IL-2 for Treg function has not been studied closely in autoimmune liver diseases The cell surface receptor for IL-2 (IL-2R) is composed of three subunits, alpha (IL-2RA, CD25), beta (IL-2RB, CD122) and common gamma (IL-2RG, CD132) All leukocytes constitutively express IL-2RG Natural Killer (NK), NK T cells (NKT) and memory CD8+ T cells also express IL-2RB and Treg constitutively express IL-2RA IL-2RA is required for high-affinity IL-2 binding, while IL-2RB and IL-2RG transduce the IL-2 signal(14) Two major signaling pathways conduct IL-2 induced responses: Signaling from IL-2RB leads to the activation of the serine/threonine kinase, AKT, and to up-regulation of anti-apoptotic molecules such as Bcl-2, which is required for T cell survival(15) Signaling from IL2RG via Jak3 leads to signal transducer and activator of transcription (STAT5) This article is protected by copyright All rights reserved Page 61 of 105 Clinical Experimental Immunology activation(16) and is needed for T cell proliferation and differentiation and expression of anti-apoptotic molecules(17, 18) Owing to their high levels of high-affinity CD25, Treg competitively consume IL-2, thereby maintaining their survival and function while suppressing bystander effector cells(19, 20) Where IL-2 availability is low, such as in the inflamed hepatic microenvironment, Treg function may be compromised and be inadequate to counteract the activated immune infiltrate A number of studies have indicated that treatment with IL-2 could improve immune-mediated diseases In rodents, type-1 diabetes mellitus could be prevented by in vivo IL-2 administration(21, 22) In humans low dose IL-2 therapy enhanced Treg frequencies and improved outcome in graft versus-host disease, vasculitis and type-1 diabetes(11, 23-25) We recently reported very low levels of IL-2 in the inflamed human liver(4) Thus, we considered that clinical grade IL-2 (Proleukin) therapy might be effective in AILD In this study we examined the effect of very low dose Proleukin (VLDP) on the biology of both peripheral and intrahepatic Treg focusing on regulatory phenotype and function Successful Treg therapy in AILD would require not only enhancing Treg phenotype and function but also recruitment of peripheral Treg to the inflamed autoimmune livers We demonstrate for the first time that VLDP selectively enhances Treg STAT5 phosphorylation and subsequently upregulates functional molecules CTLA-4, CD25, FOXP3 and Treg anti-apoptosis marker Bcl-2 in AILD VLDP also maintains liver homing chemokine receptor CXCR3 expression on Treg Importantly, VLDP enhances the suppressive function of Treg via CTLA-4 and anti-CTLA-4 can block this effect Thus, we demonstrated both phenotype and mechanistic effects of VLDP on blood and intrahepatic Treg from AILD patients suggesting that VLDP therapy may enhance immune-regulatory restoration in AILD This article is protected by copyright All rights reserved Clinical Experimental Immunology Page 62 of 105 MATERIALS AND METHODS: Ethics Statement Written informed consent was obtained from all subjects in this study Local Research Ethics Committees (LREC) and the University of Birmingham approved all experimental protocols (South Birmingham LREC reference: 98 CA/5192; Walsall LREC reference: 06/Q2708/11)) Blood and liver tissue Venous blood, collected in EDTA tubes, was obtained from healthy (control) individuals and individuals with AILD including AIH, PBC and PSC Explanted diseased liver tissue was obtained from patients undergoing liver transplantation for end-stage AILD including PBC, PSC and AIH/PBC and AIH/PSC overlap diseases Isolation of liver infiltrating leukocytes (LIL) and peripheral blood mononuclear cells (PBMC) LIL were isolated from fresh liver tissue Briefly, explanted liver tissue was diced into mm3 cubes, washed with Phosphate Buffered Saline (PBS), and then homogenised in a Seward stomacher 400 circulator (260 rpm, minutes) The homogenate was filtered through fine (63 micron) mesh (John Staniar and Co, Manchester, UK) and the lymphocytes isolated by density gradient separation using Lympholyte (VH Bio, Gateshead, UK) at 800 x g for 20 minutes The lymphocyte layer was collected and washed with PBS Cell viability was assessed by trypan blue exclusion Peripheral blood lymphocytes were likewise isolated from whole blood by density gradient separation using Lympholyte This article is protected by copyright All rights reserved Page 63 of 105 Clinical Experimental Immunology Culture of PBMC and LIL PBMC and LIL were cultured in 24-well plates at density 1x106 cells/ml in RPMI1640 with L-Glutamine medium containing Penicilin (100 IU/ml), Streptomycin (100 IU/ml), additional Glutamine (2 mM) (Gibco, California, USA) and 10% human AB serum (TCS Biosciences, Buckingham, UK) and supplemented with or IU/ml Proleukin (Aldesleukin) (Novartis, Surrey, UK) Surface phenotyping of freshly isolated intrahepatic and peripheral blood lymphocytes with or without Proleukin treatment Cell phenotypes were examined by flow cytometry Dead cells were identified by staining with the Zombie NIRTM fixable viability dye (BioLegend, San Diego, CA) or e506 viability dye (eBioscience, San Diego, CA) prior to staining with antibodies To analyse expressions of surface antigens, cells were incubated on ice for 30 minutes with antibodies against CD3, CD4, CD8, CD25, CD127 and markers of interest or isotype matched control antibodies in 2% foetal bovine serum (FBS) (Sigma Aldrich, Dorset, England) diluted in PBS After washing with 2% FBS (Sigma Aldrich), cells were fixed for 10 minutes with 3% formaldehyde solution (Sigma Aldrich) To analyse expressions of intracellular proteins, cells were fixed and stained using the FOXP3/Transcription factor staining set (eBioscience) according to manufacturer’s instructions Antibodies against surface markers (CD3, CD4, CD8 CD25, CD127) were generally added together with antibodies against intracellular markers of interest during the permeabilisation and intracellular staining step Data were acquired using a CyAN ADP flow cytometer (Dako, Glostrup, Denmark) Singlefluorophore-labelled anti-mouse IgGκ/negative control (FBS) compensation particles This article is protected by copyright All rights reserved Clinical Experimental Immunology Page 64 of 105 (BD Biosciences, Franklin Lakes, New Jersey) were used for compensation Data were analysed offline using FlowJo (Tree Star Inc., Ashland, OR) The anti-human antibodies used in flow cytometric analysis of marker expression included: anti-CD3-PeCy7 (SK7, BD Biosciences), anti-CD4-PerCP/Cy5.5 (RPA-T4, eBioscience), anti-CD4-Viogreen (VIT4, Miltenyi Biotec, Cologne, Germany), antiCD8-PE-CF594 (RPA-T8, BD Biosciences), anti-CD25-BV421 (M-A251, BD Biosciences), anti-CD45Ra-APCVio770 (T6D11, Miltenyi Biotec), anti-CD127-FITC (eBioRDR5, eBioscience), anti-CCR7-PE-CF594 (150503, BD Biosciences), antiGranzyme B-PE (GB11, eBioscience), anti-CTLA-4-PE (BN13, BD Biosciences), anti-Bcl-2-PE (100, BioLegend), anti-FOXP3-APC (PCH101, eBioscience) antiCD39-PE (A1, eBioscience), anti-TIM3-PE (F38-2E2, eBioscience), anti-OX40-PE (ACT35, BD Biosciences), anti-CD69-PE (FN50, Miltenyi Biotec), anti-2B4-PE (REA112, Miltenyi Biotec), anti-CD73-APC (AD2, eBioscience), anti-CD137-APC (4B4-1, Miltenyi Biotec), anti-GITR-APC (DT5D3, Miltenyi Biotec), anti-LAG3-APC (3DS223H, eBioscience), anti-PD-1-APC (PD1.3.1.3, Miltenyi Biotec) Analysis of STAT5 phosphorylation in response to IL-2 To examine responsiveness of PBMC or LIL to IL-2, cells in RPMI were stained with anti-CD127-FITC (eBioRDR5, eBioscience), anti-CD20-Viogreen (LT20, Miltenyi Biotec) and anti-CD56-pacific blue (HCD56, Biolegend) for 10 minutes at room temperature Cells were then stimulated for 10 minutes at 37°C with Proleukin (01000 IU/ml) Cells were fixed and permeabilised with BD Biosciences Phosflow buffers I and III according to manufacturer’s instructions then stained for hour at room temperature in 2% foetal bovine serum (Sigma) with anti-pSTAT5 (Y694)AlexaFluor 647 (47/Stat5), anti-CD3-PeCy7 (SK7), anti-CD8-PE-CF594 (RPA-T8) This article is protected by copyright All rights reserved Page 65 of 105 Clinical Experimental Immunology (all from BD Biosciences), anti-CD4-PerCPCy5.5 (RPA-T4, eBioscience) and antiCD25-PE (3G10, Miltenyi Biotec) All data were acquired using a CyAn ADP (Dako) flow cytometer and analysed using Flowjo (Tree Star) software Analysis of suppression of autologous T responder cell division by Treg in response to IL-2 CD4+CD25+CD127- Treg and CD4+CD25-CD127+ responder T cells were isolated by flow sorting following prior enrichment of total CD4+T cells from PBMC using magnetic negative selection (Biolegend) Treg were cultured overnight with or without Proleukin (5.IU/ml) Responder T cells were labelled with Cell trace violet (molecular probes, Thermofisher) and cultured overnight without stimulation Dendritic cells (DC) were derived from monocytes that were isolated from healthy donor PBMCs by magnetic negative selection (StemCell Technologies) and cultured for 5-7 days in IL-4 (500 IU/ml; Miltenyi Biotec) and GM-CSF (800 IU/ml; Berlex Laboratories, Richmond, CA) DCs and Treg were washed to remove cytokines and co-cultured for days with responder T cells in the presence of 0.5ug/ml anti-CD3 (clone OKT3, Biolegend) with or without 40ug/ml anti-CTLA-4 Division of responder T cells under 50ug/ml CTLA-4-Ig (Abatacept) was also monitored Cells were cultured at a ratio of 1DC: 20 T cells with a 1Treg: 2.5 responder T cell ratio At days, Cell trace violet dilution was measured by flow cytometry and the statistics percentage division and division index (the average number of cell divisions undergone by a cell in the original population) calculated using the Flowjo proliferation analysis platform Statistical analysis This article is protected by copyright All rights reserved Clinical Experimental Immunology Page 66 of 105 Statistical significance between two variables across multiple subsets was tested by Two-Way two-tailed ANOVA with Bonferroni post hoc analysis Significance between two populations was tested by Paired t-test and between multiple treatments by One Way ANOVA with Bonferroni post hoc analysis Analysis and graphical representation was done using GraphPad Prism version (GraphPad software, San Diego, CA) This article is protected by copyright All rights reserved Clinical Experimental Immunology Supplementary figure 4: Effect of very low dose IL-2 on expression of IL2-regulated Treg functional markers CD25, CTLA-4 and FOXP3 by CD4, CD8 and Treg cells from blood and liver PBMCs from patients with AIH, PSC and PBC and liver infiltrating lymphocytes from AILD livers were exposed to or IU/ml IL-2 (Proleukin) for 18 hours and the percentage expression of CD25, CTLA-4 and FOXP3 by CD4+, CD8+ and Treg cells examined by flow cytometry Data are mean ± SEM for donors (AIH), donors (PSC) and donors (liver) Supplementary figure 5: Sustained exposure to low dose IL-2 selectively upregulates pSTAT5 in CD4+CD25+CD127+ T cells as well as in Treg PBMCs from patients with AILD were stimulated for 18 hours with IL-2 (Proleukin) doses of 0, 1, and 10 IU/ml and expression of p(Y694)STAT5 by each leukocyte population assessed by flow cytometry (A) Percentage of each leukocyte population expressing pSTAT5 at each IL-2 dose (B) median fluorescence intensity (MFI) for pSTAT5 Supplementary figure 6: Effect of very low dose IL-2 on the suppressive ability of Treg in autoimmune liver diseases (A) CD4+CD25- T responder cells were isolated from PBMC of controls (n=3) and the effect of blocking CD28-mediated co-stimulation with Abatacept (CTLA-4-Ig) on division index at days examined (B) CD4+CD25+CD127- Treg and autologous CD4+CD25- T responder cells were isolated from PBMC of two patients with AIH (one in remission and one in relapse) T responders were labeled with Cell trace violet and following overnight exposure of Treg to or IU/ml This article is protected by copyright All rights reserved Page 104 of 105 Page 105 of 105 Clinical Experimental Immunology Proleukin were co-cultured with the Treg in the presence of anti-CD3 and dendritic cells, with or without CTLA-4 blockade Cell trace violet dilution indicating T responder cell division was analysed by flow cytometry at days This article is protected by copyright All rights reserved Page 41 of 105 Clinical Experimental Immunology 125 400 400 AIH Relapse 100 75 PSC 400 100 75 10 50 Proleukin (IU/ml) 200 100 75 10 50 200 400 600 600 800 pSTAT5 MFI 1000 800 10 50 Proleukin (IU/ml) 1000 pSTAT5 MFI Proleukin (IU/ml) 200 100 75 10 50 200 600 400 800 600 1000 PBC 1000 pSTAT5 MFI pSTAT5 MFI AIH Remission 800 10 100 Proleukin (IU/ml) Proleukin (IU/ml) (B)   1000 200 1000 100 10 0.1 25 600 50 800 75 pSTAT5 MFI 1000 100 0.01 pSTAT5 (% cells) (A)   Proleukin (IU/ml) 125 Proleukin (IU/ml) 400 Proleukin (IU/ml) 100 75 10 50 200 100 75 10 50 25 50 600 75 800 pSTAT5 MFI 1000 100 pSTAT5 (% cells) (C)   This article is protected by copyright All rights reserved B cell CD4+CD25-CD127+ CD4+CD25+CD127+ CD4+Treg CD4+CD25-CD127CD8 Cd56dim NK CD56bright NK NKT IU IL-2/ml IU IL-2/ml Clinical Experimental Immunology AIH  Blood   150 125 100 75 Treg 0.0402 50 25 CD8 0.0032 CD4 Treg 0.0254 0.0029 CD8 (B)   CD4 FOXP3 MFI FoxP3 MFI CTLA-4 MFI CD8 CD4 200 100 150 150 125 100 125 75 50 25 CD8 CD4 1500 1250 1000 750 CD8 CD4 CD8 CD4 Treg 0.0343 0.0002 100 75 50 500 400 300 200 100 CD8 1500 1250 1000 CD4 Treg 0.0038 0.0038 750 500 250 Treg CD8 CD4 CD4 Treg CD8 CD4 Treg CD8 CD4 Treg 125 100 75 50 25 0 CD8 150 25 Treg 0.0099 500 250 Treg 400 300 Treg 600 CTLA-4 MFI 100 0 1500 1250 1000 750 500 250 300 200 0.0002 500 CD25 MFI CD4 400 0.0155 600 CD25 MFI CD8 0.0066 600 500 FoxP3 MFI 200 100 CD25 MFI 0.0067 CTLA-4 MFI 0.0114 Liver   PBC  Blood   PSC  Blood   CD25 MFI CTLA-4 MFI 600 500 400 300 Page 42 of 105 1500 1250 FoxP3 MFI (A)   1000 750 500 250 Treg IU IL-2/ml IU IL-2/ml 7- g) 12 D -C 25 D C g) 750 500 250 25 12 7- g) re D -C 25 D C 7- D +C D 25 D C D 25 D C (T 12 7+ +C D D -C -C C 25 D 12 12 7- g) re 25 D D +C C D (T 12 7+ 7+ 12 D +C 25 D C C 7+ -C 25 D C re (T 712 D +C 25 D C 250 12 re 7+ 25 7+ 12 D +C 25 D C 1250 1000 FoxP3 MFI 500 D (T +C C D C g) re 712 D +C 25 D 1000 750 -C 25 D C 712 D 25 D D C 50 25 C FoxP3 MFI 25 D 12 D -C 25 25 D C CD25 MFI 75 1500 This article is protected by copyright All rights reserved C D 12 12 7+ 7- g) D (T -C 100 (T 12 D +C 25 D C P