IMMUNOLOGY

ORIGINAL ARTICLE

A peptide tetramer Tk-tPN induces tolerance of cardiac allografting by conversion of type 1 to type 2 immune responses via the Toll-like receptor 2 signal-promoted activation of the MCP1 gene Zuoqing Li,1,* Neng Yang,2,* Ling Zhou,1 Peng Gu,2 Hui Wang,3 Yun Zhou,1 Peijun Zhou,3 Liming Lu1 and Kuang-Yen Chou2 1

Shanghai Institute of Immunology, Shanghai, Department of Immunology, Shanghai Jiaotong University School of Medicine, Shanghai, and 3Transplantation Unit, Shanghai Ruijin Hospital, Shanghai, China 2

doi:10.1111/imm.12569 Received 29 September 2015; revised 24 November 2015; accepted 16 December 2015. *These authors contributed equally to this work. Correspondence: Kuang-Yen Chou, Department of Immunology, Shanghai Jiaotong University School of Medicine, 280 South Chongqing Road, Shanghai 200025, China. Email: [email protected] and Liming Lu, Shanghai Institute of Immunology, 280 South Chongqing Rd, Shanghai 200025, China. Email: [email protected] Senior author: Kuang-Yen Chou

Summary The plant protein trichosanthin (Tk) and its derived peptide tetramer TktPN have been shown to stimulate the type 2 immune responses for treating autoimmune disease. This work explores the possibility of using Tk-tPN as a non-toxic immunosuppressant to induce transplantation tolerance using the mechanisms by which T-cell-mediated immune responses are transferred from type 1 to type 2 through innate immunity-related pathways. Immunocytes and cytokine secretions involved in the mouse cardiac allografting model with Tk-tPN treatment were characterized. Identification of critical genes and analysis of their functions through Toll-like receptor (TLR) -initiated signalling and the possible epigenetic changes were performed. Mean survival times of the cardiac allografts were delayed from 7
7  0
3 days (control) to 22
7  3
9 days (P < 0
01) or 79
1  19
2 days (P < 0
0001) when Tk-tPN was introduced into the recipients alone or together with rapamycin, respectively. The grafting tolerance was donor-specific. The secretion pattern of the type 1 cytokine/ transcription factor (IL-2+ IFN-c+ T-bet+), which is responsible for the acute graft rejection, was shifted to the type 2 factor (IL4+ IL-10+ Gata3+), together with a selective expansion of the IL-4/IL-10producing CD8+ CD28 regulatory T-cell subset. A TLR2-initiated high expression of chemokine gene MCP1 was detectable simultaneously. Epigenetically Tk/Tk-tPN could also acetylate the histone H3K9 of MCP1 promoter to skew the immunity towards T helper type 2 responses. Tk/ Tk-tPN is therefore capable of down-regulating the type 1 response-dominant rejection of cardiac allografts by evoking type 2 immunity through the activation of a TLR2-initiated signalling pathway and MCP1 gene to expand the IL-4/IL-10-secreting CD8+ CD28 regulatory T cells. Tk-tPN could be a promising novel immunosuppressant to induce tolerance in allotransplantation. Keywords: cardiac allografting; CD8+ CD28 regulatory T cells; monocyte chemoattractant protein-1; Toll-like receptor 2 signalling; trichosanthinderived peptide

Abbreviations: Ana-1, macrophage cell line from a C57BL/6 mouse; IFN, interferon; IL, interleukin; KO, knockout; mAb, monoclonal antibody; MCP-1, monocyte chemoattractant protein-1; MLC, mixed lymphocyte culture; MLR, mixed lymphocyte reaction; MST, mean survival time; NLR, NOD-like receptor; PEM, peritoneal macrophage; Raw (Raw284.7), macrophage cell line from a BALB/c mouse; Rapa, rapamycin; Th1, T helper type 1; Tk, trichosanthin; Tk-tPN, tetramer of a Tk-derived peptide; TLR, Toll-like receptor; Treg, regulatory T; urP, unrelated peptide; WT, wild-type ª 2015 John Wiley & Sons Ltd, Immunology, 147, 355–366

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Z. Li et al. Introduction Based on differential expression patterns of cytokines/ transcription factors and biological functions, T-cellrelated immune responses divide into three categories: type 1 (IL-2+ IFN-c+ T-bet+), type 2 (IL-4+ IL10+ Gata3+) and type 3 (IL-17+ IL-22+ RORcT+).1,2 Although their prototypes are CD4 T helper type 1 (Th1) -mediated, CD4 Th2-mediated and CD4 Th17-mediated responses, respectively, broader ranges of reactions have been suggested. For example, CD8 Tc1-initiated and CD8 cytotoxic T lymphocyte-initiated responses are included within the type 1, whereas CD8 Tc2-, CD4 Th9- and CD4 follicular T helper-mediated responses are more connected with the type 2 response. Besides, the Th17 subset is sometimes discussed together with Th1 cells because of their functional similarity and their phenotypic plasticity under the influence of certain cytokines. Importantly, most immune-related reactions in clinical diseases can be conventionally identified as being predominantly type 1 or type 2 responses, or both. Among them, for example, immune reactions involved in anti-virus, antitumour and anti-graft rejection are included in the type 1 responses. On the other hand, antibody production, asthma and anti-worm are induced by the type 2 reactions. An urgent problem is how to manipulate and regulate the two main types of responses in connection with diseases. A group of factors and agents that are active in innate immunity have been delineated so far with their attributions of initiating the adaptive type 1/type 2 responses. Among them are Toll-like receptors (TLRs),3 NOD-like receptors (NLRs),4 cytokines/chemokines,5 transcription factors,6 Notch signals,7 cd T cells8 and dendritic cells,9 as well as various immunological preparations.10 Recently, a range of innate lymphoid cells was identified that were capable of dictating lymphocyte differentiation and regulating both innate and adapted immune responses.11 The group 2 innate lymphoid cells, for example, are active in the instigation of Th2 polarization by secretion of the type 2 cytokines interleukin-4 (IL-4), IL-5 and IL-13.12,13 Trichosanthin (Tk), a 247-amino-acid linear peptide isolated from a medicinal herb Trichosanthes kirilowii Max, shows stable immunobiological activity at physiological concentrations (≤ 1 lg/ml); it induces a CD8 Tcell-governing type 2 immune response with low interferon-c (IFN-c) secretion and abundant production of IL-4 and IL-10.14–17 The underlying mechanism also relies upon a pattern shift of cytokine production and transcription factor expression from IL-2+ IFN-c+ T-bet+ to IL-4+ IL-10+ Gata3+, resulting in a transfer from type 1 immunity to the type 2 response. A Tk-derived peptide tetramer Tk-tPN was prepared to show the property of inducing suppression that is free from toxicity in terms

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of cytolysis and apoptosis.18 Tk and Tk-tPN are also capable of activating the CD8+ CD28 regulatory T (Treg) cells.18,19 This paper deals with the ability of Tk-tPN to delay the rejection of cardiac allografts by conversion of the type 1 response to the type 2 by selectively activating monocyte chemoattractant protein-1 (MCP-1). Activation of the MCP-1-encoding gene is dependent on a signalling pathway initiated by TLR2. Tk/Tk-tPN is therefore promising as a reagent in clinics for down-regulating the type 1 reaction to induce transplantation tolerance.

Materials and methods Animals Male BALB/c (H-2d), C57BL/6 (H-2b), C3H/He (H-2k) mice aged 8–12 weeks were supplied by the Sino-British Sipper/BK Animal Laboratory (Shanghai, China) and used in accordance with the regulations of the Scientific Investigation Board of the Shanghai Jiaotong University School of Medicine.

Cell preparations and lymphoproliferation Splenocytes and lymph node mononuclear cells were routinely prepared. The fractionated T cells with purity > 95% were first isolated by centrifugation with mouse Ficoll solution and then incubated with immunobeads (Miltenyi Biotech, Bergisch Gladbach, Germany). CD4and CD8-enriched T-cell subsets were prepared by depleting the CD8- or CD4-positive cells from the T-cell population with monoclonal antibodies (mAbs). For mixed lymphocyte culture (MLC), fractionated CD4+ T cells were used as responding cells (R) and the 25 Gy-irradiated allogeneic splenocytes were used as stimulating cells (S). Equal numbers of R and S cells (2 9 105) were mixed in the wells of round-bottom 96-well microplates. Two hundred microlitres of RPMI-1640 culture media (Gibco, Grand Island, NY) supplemented with 10% fetal calf serum (Hyclone, Logan, UT), 25 mM HEPES, 5 9 105 mol 2-mercaptoethanol, 100 U/ml penicillin and 100 lg/ml streptomycin were added to each well. The cells were cultured at 37° and 5% CO2 for 6 days. Sixteen hours before harvesting of the cultures, 1 lCi/well of [3H] thymidine ([3H]TdR; Institute of Atomic Nucleus, Chinese Academy of Sciences, Shanghai, China) was added to each well. The cells were then harvested to determine the radioactivity with a liquid scintillation counter (MicroBeta TriLux, Turku, Finland). The results are expressed as the mean counts per minute (cpm)  SD of the triplicates. The suppression effects of Tk/Tk-tPN or the added syngeneic modulating cells on mixed lymphocyte reaction (MLR) are expressed as repressed relative responses (RR) or % suppression (100– RR).

ª 2015 John Wiley & Sons Ltd, Immunology, 147, 355–366

Peptide Tk-tPN induces grafting tolerance by MCP-1-promoted type 2 response RR ð%Þ ¼

cpm of exp combination with Tk  100 cpm of control without Tk

Some MLR was also determined by carboxyfluorescein diacetate succinimidyl easter (CFSE) assay, in which stimulating cells were treated with mitomycin C (40 lg/ml, 37° for 1 hr) and responding T cells were stained with CFSE at dose 5 mM. The results were determined by flow cytometry.

Trichosanthin-derived peptide Trichosanthin was available as a crystalline water solution from Shanghai Jin-Shan Pharmaceutical Co. (Shanghai, China). The Tk stock solution was diluted to 0
05 mg/ml with saline and sterilized using a 0
2-lm filter. Twenty-four overlapping peptides, each of which was 15 amino acids long and suitable for processing and presentation by MHC class II molecules in antigen-presenting cells, were synthesized by GL Biochem Peptide Ltd (Shanghai, China) to cover the entire Tk molecule. According to a differential ability to repress the ovalbumin-specific lymphoproliferation in two mouse strains (C57BL/6 and C3H.He), which have been identified as high susceptibility (HS) and low susceptibility (LS) to Tk-induced hyporeaction, respectively,19 those peptides with strong suppressive activity but no cytotoxic activity were selected. One of them was further conjugated to form a Tk-tPN tetramer with a molecular weight of 13 700 and a purity of > 90%.18 The cytotoxicity and apoptosis of the tetramer have been carefully excluded through Trypan blue exclusion test (for cell viability) and by analysis with Annexin-V/propidium iodide kits (for apoptosis).

Flow cytometry The surface markers of lymph node mononuclear cells and T cells were stained with the FITC-conjugated rat anti-mouse CD4, CD8 and CD25 antibodies (eBioscience, San Diego, CA) or the phycoerythrin-conjugated rat antimouse CD28 antibody. Cells were washed, fixed and assayed to determine the percentages of cell subsets with the antibodies by a FACSCaliburTM flow cytometer (Becton Dickinson, Franklin Lakes, NJ). The gating strategy for designation of the CD8+ CD28 T-cell population was based on the fluorescence minus one control to divide the cells into CD3+ CD8+ CD28+ and CD3+ CD8+ T-cell populations. The latter was treated as CD28 negativity. For identification of CD4+ CD25+ Foxp3+ Treg cells, the surface CD markers were routinely stained. The intranuclear staining for transcriptional factor Foxp3 was performed with phycoerythrin-conjugated anti-Foxp3 antibody (Santa Cruz Biotechnology, Dallas, TX), Permeª 2015 John Wiley & Sons Ltd, Immunology, 147, 355–366

abilization Buffer and Staining Buffer Set (eBioscience). The Treg subset was then analysed by flow cytometry.

Cardiac allotransplantation According to Hancock et al.,20 heterotopic abdominal cardiac allografting was performed with C57BL/6 (B6) mice as donors and BALB/c mice as recipients. The recipients were treated with 1 mg Tk-tPN/kg/day by intravenous injection before the grafting procedure and by intraperitoneal infusion after grafting. Rapamycin (Wyeth Pharmaceutical, Madison, NJ) at a concentration of 1 mg/kg was also orally introduced as a positive control from postoperative day 0 to day 14. Cardiac graft survival was evaluated by daily palpation. The cessation of beating was interpreted as rejection. The mortality rate due to surgical or technical failure of the transplant procedure was < 5% in our experiments.

Real-time RT-PCR) To probe gene expression, RNA was extracted from cells or tissues with TRIzol reagent (Invitrogen Life Technologies, Carlsbad, CA). The tissues of the heart allografts were harvested, minced and incubated in RPMI-1640 medium containing 0
2 mg/ml collagenase and 0
2 mg/ml DNase I (Sigma, St Louis, MO). The RNA samples (2 lg) were reverse transcribed with a thermocycler (Roche Applied Science, Indianapolis, IN) and SYBR Green dye. The 2DDCT method was used to calculate fold changes.21 Relative expression levels were plotted as the ratio of the target gene versus the housekeeping gene Hprt (hypoxanthine-guanine phosphoribosyltransferase). PCR amplification was routinely performed with the synthesized sense/ antisense primers of target genes. They include several groups: (i) the genes coding for essential cytokines and transcriptional factors by which the type 1/type 2 immune responses can be categorized: IFN-c, IL-2, IL-4, IL-10, transforming growth factor-b, T-bet and Gata3; (ii) 31 macrophage-related genes, which are listed in Fig. 3(b), to reveal the expression profile after Tk treatment; (iii) the genes coding for the essential components to distinguish the macrophage subpopulations M1 and M2: inducible nitric oxide synthase, arginase-1, IL-10 and IL-12; and (iv) other genes including MCP1, TLR2 and Foxp3. The experiments were usually repeated three times and representative data are displayed.

TLR2-initiated signalling in Tk-induced responses A Tk-glutathione S-transferase (GST) fusion gene was constructed and cloned using the plasmid pGEX-4T-3/Tk, in which the vehicle pGEX-4T-3 plasmid (Pharmacia, Uppsala, Sweden) was ligated with a 741-bp mat Tk peptide.22 The soluble Tk-GST fusion protein was expressed 357

Z. Li et al. and purified by glutathione–Sepharose 4B affinity chromatography and identified by 10% SDS–PAGE. An electrophoresis mobility shift assay was performed to investigate the binding of transcription factors and the co-precipitation of Tk-GST with TLR2. The binding of TLR2 with Tk in antigen-presenting cell lysates was assayed according to the Promega GST pull-down protocol. The increased activation and/or translocation of phosphorylated nuclear factor-jB, activator protein-1 and mitogen-activated protein kinases due to TLR2-initiated signal transduction was further determined with the relevant mAbs (Cell Signaling Technology, Danvers, MA).

tion.18 Besides, Tk/Tk-tPN was also able to expand the CD8+ CD28 T-cell subset in an anti-CD3 mAb-initiated lymphocyte reaction (Fig. 1e), the pattern of which was the same as those in our previous studies. Identical or comparable results were recorded both for treatment with Tk (in a low dose range 0
01–1 lg/ml) and with Tk-tPN (dose range 2–20 lg/ml) in their stimulation of suppression (Fig. 1a,b) and in their activation of the CD8 Treg cells (Fig. 1e), so Tk and Tk-tPN are treated as the same immunosuppressant in our clinical and mechanism studies. Repeated examinations excluded any cytotoxicity of cytolysis and apoptosis both for Tk and Tk-tPN in the dose range we adopted.17–19

Determination of H3K9 acetylation levels Using the Eukaryotic Promoter Database (EPD) as a reference, a 1288-bp key sequence of the MCP1 promoter region was amplified from RAW264.7 mouse cells. After constructing the sequence-containing reporter pGL3/ MCP1p, the methylation of the CpG loci within the MCP1 promoter and the acetylation of histone 3 lysine 9 (H3K9) were determined by gene transfection with the Nucleofactor Kit V for the RAW 264.7 cell line (Lonza, Basel, Switzerland) together with a bisulphite sequencing PCR and chromatin immunoprecipitation with the SimpleChIPTM Enzymatic Chromatin IP Kit (magnetic beads) (Cell Signaling Technology).

Statistical analysis Intergroup comparisons were made using two-tailed Student’s t-test and the one-way analysis of variance. A P-value < 0
05 was considered statistically significant.

Results Tk/Tk-tPN induces suppression of MLR via conversion of type 1 to type 2 immune responses About 90% of one-way MLRs were suppressed when native Tk or its peptide tetramer Tk-tPN was added into cell cultures at dosages 0
5 lg/ml and 10 lg/ml, respectively, as determined by [3H]TdR uptake assay (Fig. 1a). Similar results were observed when the suppression was assayed by CFSE staining (Fig. 1b). This was accompanied by a dramatic change of the cytokine secretion levels, i.e. the repressed IL-2/IFN-c and the increased IL-4/IL-10 (Fig. 1c), together with decreased and increased expression of transcription factor T-bet and Gata3, respectively (Fig. 1d). The changes suggest a phenotypic alteration of immune response from type 1 (IL-2+ IFN-c+ T-bet+) to type 2 (IL-4+ IL-10+ Gata3+) after Tk/Tk-tPN was introduced as a suppression inducer, which confirmed our previous observations on the down-regulatory activity of Tk/Tk-tPN for the ovalbumin-induced lymphoprolifera358

Tk-tPN prolongs the survival of cardiac allografts A murine cardiac allograft model was heterotopically established. As shown in our two independent experiments (Fig. 2a,b), the Tk-tPN treatment of BALB/c recipients transplanted with C57BL/6 hearts obviously extended the survival of the grafts in which the oral infusion of rapamycin was routinely used as a positive control. The mean survival times (MSTs) for the treatments with Tk-tPN and rapamycin alone in experiment 2 were 22
7  3
9 and 21
6  3
0 days versus 7
7  0
3 days (ctrl) (P < 0
01), respectively (Fig. 2b). The most profound suppressive effects were recorded when Tk-tPN was introduced together with rapamycin, yielding an MST of 79
1  19
2 days (P < 0
0001). There was no significant difference (P > 0
05) between this MST and that of the syngeneic grafting (81
6  10
2 days), which was set as negative control.

Immunobiological traits of the Tk-tPN-induced grafting tolerance As indicated in our in vitro experiments (Fig. 2c), the TktPN-induced immune suppression only appeared when the donor cells in the MLR were from the original B6 mice instead of the other (C3H/He) mouse strain, suggesting a donor-specific suppression of the allo-reaction in our experiments. An examination of cytokine gene expression on day 7 after transplantation both in heart grafts and in the recipient spleens indicated that the type-1-related cytokines either remained unchanged (IL-2) or decreased after treatment with Tk-tPN and/or rapamycin (Fig. 2d). The production of type-2-related cytokines IL-4, IL-10 and transforming growth factor-b showed a remarkably increase, and the most profound changes were recorded in the double treatment by TktPN plus rapamycin. This predominant expression of type 2 cytokines in the recipients implies onset of the conversion for immune responses from type 1 to type 2 after Tk-tPN treatment. ª 2015 John Wiley & Sons Ltd, Immunology, 147, 355–366

Peptide Tk-tPN induces grafting tolerance by MCP-1-promoted type 2 response R+S+Tk

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[3H]TdR uptake (cpm×103)

[3H]TdR uptake (cpm×103)

(a)

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R+S (MLR) R+S+Tk (0·1^) R+S+Tk-tPN (5^)

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CD28

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103

103

103

102

102

102

101

101

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150

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Ck level (pg/ml)

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104

^µg/ml, *P