γδ T Cell Receptor Deficiency Attenuated Cardiac Allograft Vasculopathy and Promoted Regulatory T cell Expansion

Authors

  • H. Zhu,

    Corresponding author
    • Department of Anesthesiology and The State Key Laboratory Breeding Base of Basic Science of Stomatology (Hubei-MOST) & Key Laboratory of Oral Biomedicine Ministry of Education, School & Hospital of Stomatology, Wuhan University, Wuhan, China
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  • J. Li,

    1. Department of Oncology, Wuhan Central Hospital, Wuhan, China
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  • S. Wang,

    1. Department of Cardiovascular Surgery, Union Hospital, Huazhong University of Science and Technology, Wuhan, China
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  • K. Liu,

    1. Department of Anesthesiology and The State Key Laboratory Breeding Base of Basic Science of Stomatology (Hubei-MOST) & Key Laboratory of Oral Biomedicine Ministry of Education, School & Hospital of Stomatology, Wuhan University, Wuhan, China
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  • L. Wang,

    1. Department of Anesthesiology and The State Key Laboratory Breeding Base of Basic Science of Stomatology (Hubei-MOST) & Key Laboratory of Oral Biomedicine Ministry of Education, School & Hospital of Stomatology, Wuhan University, Wuhan, China
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  • L. Huang

    1. Department of Anesthesiology and The State Key Laboratory Breeding Base of Basic Science of Stomatology (Hubei-MOST) & Key Laboratory of Oral Biomedicine Ministry of Education, School & Hospital of Stomatology, Wuhan University, Wuhan, China
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  • Jun Li and Hongfei Zhu contributed equally to this paper.

Correspondence to: H. Zhu, Department of Anesthesiology and The State Key Laboratory Breeding Base of Basic Science of Stomatology (Hubei-MOST) & Key Laboratory of Oral Biomedicine Ministry of Education, School & Hospital of Stomatology, Wuhan University, Luoyu Road 237#, Wuhan 430079, China. E-mail: hongfeizhu1973@163.com

Abstract

γδ T cell comprises about 5% of the overall T cell population, and they differ from conventional αβ T cells. Previous studies have indicated the contribution of γδ T cell to acute allograft rejection, but the role of γδ T cell in cardiac allograft vasculopathy (CAV) is not investigated. Hearts of adult B6.C-H-2bm12KhEg were heterotopically transplanted into major histocompatibility complex (MHC) class II-mismatched C57BL/6 mice (wild-type, γδ TCR−/−), which is an established murine model of chronic allograft rejection without immunosuppression. The survival of grafts was monitored daily by abdominal palpation until the complete cessation of cardiac contractility. Our current study demonstrated that γδ T cell receptor (TCR) deficiency significantly attenuated CAV, and this effect coincides with low expression of Hmgb1, IFN-γ and IL-17 while increased number of CD4+CD25+Foxp3+ regulatory T cells, and depletion of regulatory T cells abrogated the prolonged allograft survival induced by γδ TCR deficiency. γδ TCR deficiency resulted in attenuated CAV and prolonged graft survival in murine models of cardiac transplantation, and this effect was associated with enhanced expansion of regulatory T cells.

Introduction

Cardiac transplantation is the last resort for patients with end-stage heart failure. Short-term patient survival of acute rejection has been substantially improved over the past years, but long-term survival has not been dramatically raised so far. The predominant obstacle has been cardiac allograft vasculopathy (CAV) [1]. Hence, research effort has been directed at exploring strategies that can overcome the shortcoming of conventional immunosuppressive to effectively inhibit the development of CAV.

γδ T cell comprises about 5% of the overall T cell population, and they differ from conventional αβ T cells in that they express invariant γ and δ chains as part of their T cell receptor [2]. γδ T cells appear to lack the requirement for conventional antigen presentation, and this has contributed to the hypothesis that these cells act as tissue-resident immune sentinel cells [3]. However, the fundamental roles of γδ T cells in transplant rejection and tolerance have not been well documented. Previous studies indicated that γδ T cell showing protective functions in human kidney and liver graft [4, 5]. Also, some investigators reported that γδ T cell contributed to development of transplant rejection [6, 7]. Our previous study showed that γδ T cells represented a dominant IL-17-producing lymphocyte subset in cardiac transplantation [8]. Importantly, recent report by Naoyuki Kimura demonstrated that recipient γδ T cells accelerate acute allograft rejection in major histocompatibility complex (MHC) full mismatch murine heart transplant model [9]. But, the role of γδ T cell in chronic cardiac transplant rejection has not been investigated.

In the present work, we investigate the role of γδ T cells in CAV utilizing MHC-II mismatched murine heart transplantation models. The result showed that deficiency of γδ T Cell Receptor (TCR) resulted in attenuated CAV in murine models of cardiac transplantation. This effect coincides with low expression of Hmgb1, IFN-γ and IL-17 while increased expansion of CD4+CD25+Foxp3+ regulatory T cells, and depletion of regulatory T cells abrogated the prolonged allograft survival induced by γδ TCR deficiency.

Materials and methods

Animals

C57BL/6 (H−2b) mice were considered as wild-type (WT) and were purchased from the Institute of Laboratory Animal Sciences of Chinese Academy of Medical Sciences (Beijing, China). C57BL/6 (H−2b) γδTCR-deficient mice and B6.C-H-2bm12KhEg (H-2bm12) mice were purchase from Jackson Laboratory (Bar Harbor, ME, USA). Both C57BL/6 and B6.C-H-2bm12KhEg mice are genetically, but differ in their expression of the MHC class II molecule, I-Ab. All the mice were male at 8 weeks of age and 25–30 g in weight, which were housed in specific pathogen-free facility with regular food and water adlibitum. Experiments were approved by the Institutional Animal Care and Use Committee at Tongji Medical College (Wuhan, China).

Heterotopic cardiac transplant and post-transplant therapies

Heterotopic cardiac transplantation was performed using a procedure described by Corry et al. [10]. Briefly, cardiac allografts were transplanted in the abdominal cavity by anastomosing the aorta and pulmonary artery of the graft end-to-side to the recipient's aorta and vena cava, respectively. Hearts of adult B6.C-H-2bm12KhEg were heterotopically transplanted into MHC class II-mismatched C57BL/6 mice (WT, γδ TCR−/−), which is an established murine model of chronic allograft rejection without immunosuppression. The strength and quality of cardiac impulses were graded by palpation on daily basis. In γδ TCR−/− group (n = 12), six animals were sacrificed on day 65 post-transplantation. In anti-γδTCR mAb-treated group (n = 12), recipients were administrated by intraperitoneal injection with 500 μg anti-γδTCR mAb (Pharmingen, San Diego, CA, USA) twice weekly for 6 weeks [11]. And six animals were also sacrificed on day 65 post-transplantation. In WT group (n = 6), cardiac allografts were obtained when rejection occurrence. For regulatory T cell depletion, recipients were intraperitoneally injected with 500 μg of anti-mouse CD25 mAb (PC61; Bioexpress Cell culture, Kaysville, UT, USA) 1 day before transplantation [12].

Histopathology and immunohistochemistry

Cardiac allograft tissues were stained with elastic-van Garson's (EvG) stains [13]. To evaluate CAV, three different sections were observed, and only vessels exceeding 80 μm in diameter were included; the area encompassed by the lumen and internal elastic lamina was analysed with computer-based software (optimas, Berlin, Germany); the luminal occlusion rate was calculated by the following formula: Luminal occlusion rate = (Internal elastic lamina area − luminal area)/Internal elastic lamina area. Data were analysed for the severity of CAV, as recently described in detail [14]. Immunohistochemical staining for CD4, CD8 and CD68 was performed as previously described [15]. Slides for immunofluorescence staining with Abs specific for CD4, Foxp3 and IL-17 (BD Bioscience, San Jose, CA, USA) were fixed with acetone and blocked serially with 5% donkey serum, clarified Moffat dried milk and Fc blocker (Accurate Chemical, Westbury, NY, USA). After washing, the slides were incubated with primary Ab, washed again, incubated with donkey anti-rat IgG conjugated to either fluorescein isothiocyanate (FITC) (green fluorescence) or CY3 (red fluorescence). After washing, slides were viewed under an epifluorescent microscope (DMR; Leica Microsystems, Wetzlar, Germany). Binding specificity was determined using an isotype-matched Ab. To evaluate cell infiltration, five fields were randomly selected from one section to count the number of positive cells in each field.

Fluorescence-activated cell sorter (FACS) analysis

Accordingly previously study reported by Gorbacheva et al. [16], infiltrated cells in the allograft were isolated. Cells were stimulated with 0.1 μg/ml PMA (Sigma, St Louis, MO, USA), 1 μg/ml ionomycin (Sigma) and Brefeldin A (10 μl/6 ml; eBioscience, San Diego, CA, USA) for 4 h at 37 degrees. Flow cytometry was performed on a FACSCalibur (BD Immunocytometry Systems, San Jose, CA, USA) according to the manufacturer's instructions, and data were analysed using CellQuest software (BD eBioscience, San Diego, CA, USA). Isotype controls were given to enable correct compensation and confirm antibody specificity.

SYBR green real-time RT-PCR

The recipient mice were killed at indicated time after transplantation. Cardiac allografts were collected and subjected to RNA isolation using the TRIzol (Invitrogen, Carlsbad, CA, USA) reagent according to the manufacturer's instruction. cDNA was synthesized from 2 μg RNA using a first-strand DNA synthesis kit (Fermentas Life Sciences, St Leon-Rot, Germany). The mRNA levels for HMGB1, IFN-γ, IL-17 and Foxp3 in the grafts were analysed by real-time PCR using iCycler (Bio-Rad, Hercules, CA, USA). PCR reaction mixture was prepared using SYBR Premix Ex Taq (TaKaRa, Otsu, Shiga, Japan) according to the manufacturer's instructions. Primers for PCR amplification were used as previously described [17] (Supplementary digital content, Table 1). Relative expression levels for cytokines were normalized by GAPDH and calculated using the inline image method and expressed in arbitrary units. The expression of each cytokine in normal mice was used as calibrator.

Table 1. The primer sequences used in this study
GenePrimer sequences
Hmgb1Forward: 5′-GCGGACAAGGCCCGTTA-3′
Reverse: 5′-AGAGGAAGAAGGCCGAAGGA-3′
IFN-γForward: 5′-AGCGGCTGACTGAACTCAGATTGTAG-3′
Reverse: 5′-GTCACAGTTTTCAGCTGTATAGGG-3′
IL-17Forward: 5′-GACCAGGATCTCTTGCTGGA-3′
Reverse: 5′-GGACTCTCCACCGCAATGA-3′
Foxp3Forward: 5′-TACTTCAAGTTCCACAACATGCGACC-3′
Reverse: 5′-CGCACAAAGCACTTGTGCAGACTCAG-3′
GAPDHForward: 5′-TTCACCACCATGGAGAAGGC-3′
Reverse: 5′-GGCATGGACTGTGGTCATGA-3′
Statistical analysis

Data are expressed as mean ± SEM. Kaplan–Meier methods were used to calculate for the survival of grafts. The difference among groups was performed by one-way analysis of variance followed by Bonferroni correction. Between two groups of mice, an unpaired Student's t-test was performed to determine statistical significance. P value < 0.05 was considered to be statistically significant.

Results

Survival time and histopathological analysis of cardiac allograft

We examined the impact of γδ TCR on heart allograft survival. γδ TCR deficiency resulted in long-term survival of allograft [median survival time (MST) = 90 days]. In contrast, allograft in WT recipients showed shorter survival of allograft (MST = 65 days, P < 0.005 as compared with γδ TCR−/− group or anti-γδTCR mAb-treated group; Fig. 1A). On day 65 post-grading, the vessels in cardiac allografts from WT group were almost obstructed (mean CAV grade: 2.58 ± 0.14) and from γδ TCR−/− group or anti-γδTCR mAb-treated group were slightly affected (mean CAV grade in γδ TCR−/− group: 1.16 ± 0.11; mean CAV grade in anti-γδTCR mAb-treated group: 1.41 ± 0.14, P < 0.001) (Fig. 1B).

Figure 1.

(A) The survival of heart allografts in γδ TCR−/− recipient or anti-γδTCR mAb-treated group was significantly prolonged to 90 days (P < 0.005 versus wild-type groups, MST = 65 days), and heart allografts survival in γδ TCR−/−+ anti-CD25 mAb or anti-γδ TCR mAb+anti-CD25 mAb group was reduced to 60 or 62 days; (B) Indicating the cardiac allograft vasculopathy grade of cardiac allografts on day 65 post-transplantation.

Analysis of leucocyte infiltration and Hmgb1 and IFN-γ expression in cardiac allograft

We examined the number of graft-infiltrating immune cells. On day 65 post-grading, the number of graft-infiltrating CD4+ T, CD8+ T cells and CD68+ macrophages was markedly lower in γδ TCR−/− group or anti-γδTCR mAb-treated group (Fig. 2A). The expression of Hmgb1 and IFN-γ in allograft from γδ TCR−/− group or anti-γδTCR mAb-treated group was also lower as compared from WT group (Fig. 2B).

Figure 2.

Analysis of cells infiltration in cardiac allografts on day 65 post-transplantation. (A) Indicating the number of CD4+ T, CD8+ T cells and CD68+ macrophages in cardiac allograft; (B) Gene expression of Hmgb1 and IFN-γ in cardiac allograft; Asterisks on the top of an error bar indicate statistically significant differences between γδ TCR−/− or anti-γδTCR mAb-treated group and wild-type group. P < 0.05.

Analysis of IL-17 and Foxp3 expression in cardiac allograft

First, we examined IL-17 expression in allograft. The results showed that there was lower IL-17+ cell infiltration and IL-17 mRNA expression in γδ TCR−/− group or anti-γδTCR mAb-treated group (Fig. 3A–C). Then, we detected Foxp3 expression in allograft, and we found that both the number of CD4+Foxp3+ regulatory T cells and the expression of Foxp3 mRNA were significantly increased in γδ TCR−/− group or anti-γδTCR mAb-treated group as compared with WT group (Fig. 4A–C). Also, the proportion of CD25+Foxp3+ regulatory T cells in CD4+ T cells from graft-infiltrating cells was significantly increased in γδ TCR−/− group [(8.54 ± 1.11)%] or anti-γδTCR mAb-treated group [(7.87 ± 0.95)%] as compared with WT group [(0.92 ± 0.18)%, < 0.005] (Fig. 4D–E).

Figure 3.

(A) Immunofluorescence staining for IL-17 on day 65 post-transplantation was shown (×400), red fluorescence indicate IL-17 positive; (B) Indicating the number of positive IL-17+ cells on day 65 post-transplantation; (C) Gene expression of IL-17 in cardiac allografts on day 65 post-transplantation; Asterisks on the top of an error bar indicate statistically significant differences between γδ TCR−/− or anti-γδ TCR mAb-treated group and wild-type group. P < 0.005.

Figure 4.

(A) Immunofluorescence staining for Foxp3 on day 65 post-transplantation was shown (×400), green fluorescence indicate CD4 positive, and red fluorescence indicate Foxp3 positive; (B) Indicating the number of positive CD4+Foxp3+ cells in cardiac allograft on day 65 post-transplantation; (C) Gene expression of Foxp3 in cardiac allograft on day 65 post-transplantation; (D) fluorescence-activated cell sorter (FACS) for regulatory T cell in allograft on day 65 post-transplantation; (E) Indicating the proportion of CD25+Foxp3+ regulatory T cells in CD4+ T cells from graft-infiltrating lymphocytes; Asterisks on the top of an error bar indicate statistically significant differences between γδ TCR−/− or anti-γδ TCR mAb-treated group and wild-type group. P < 0.005.

Prolonged allograft survival is dependent on enhanced regulatory T cell expansion

Regulatory T cells could suppress the mechanism of both acute and chronic rejection and, therefore, prolonged allograft survival. We also found that the number of CD4+CD25+Foxp3+ regulatory T cells was increased in the allograft with γδ TCR deficiency. To further investigate the function of regulatory T cells in transplant rejection, we injected anti-CD25 mAb into allograft recipients with γδ TCR deficiency and found that the prolonged survival time was abrogated with regulatory T cell depletion (P < 0.05 as compared with γδ TCR−/− group or anti-γδTCR mAb-treated group, MST = 60 days or 62 days) (Fig. 1A). And the vessels in cardiac allografts with γδ TCR deficiency in combination with regulatory T cell depletion were almost obstructed (mean CAV grade in γδ TCR−/−+ anti-CD25 mAb: 2.83 ± 0.11; mean CAV grade in anti-γδ TCR mAb+anti-CD25 mAb group: 2.75 ± 0.13) (Fig. 1B).

Discussion

Our current study demonstrated that γδ TCR deficiency significantly attenuated CAV, and this effect coincides with low expression of Hmgb1, IFN-γ and IL-17 while increased number of CD4+CD25+Foxp3+ regulatory T cells, and depletion of regulatory T cells abrogated the prolonged allograft survival induced by γδ TCR deficiency.

The fate of a transplanted organ is determined partly by the number of induced effector T cells. The effector T cell pool size is, in turn, dependent on several factors such as precursor frequency, factors involved in Ag presentation and costimulation, proinflammatory signals produced by innate immune system [18]. Cells of the macrophage lineage are a major component of the infiltrate in allografts undergoing T cell-mediated rejection [19]. Macrophages are involved in the innate and adaptive immunity during allograft rejection playing a key role in the initiation and effector phases of the immune response [20, 21]. On the basis of previous findings, our experiment further proved that decreased number of effecter T lymphocyte and macrophages infiltration may contribute to reduce CAV by γδ TCR deficiency.

High-mobility group box 1 (HMGB1) was originally characterized as a nuclear protein implicated in facilitating DNA binding [22]. Recent studies have now consistently demonstrated that it also functions as a critical mediator to initiate innate immune response on inflammatory or other insults. HMGB1 is also evident in the pathogenesis of some autoimmune disorders characterized by altered Th17 responses such as multiple sclerosis, rheumatoid arthritis and experimental autoimmune encephalomyelitis [23]. Importantly, previous studies have demonstrated a role for HMGB1 in the initiation and progression of allograft rejection [17, 24]. Another proinflammatory cytokine INF-γ plays a central role in acute allograft rejection [25, 26]. Although the exact pathogenesis of CAV remains to be established, previous studies also suggested the pivotal role of INF-γ in triggering the pathological changes associated with CAV [27, 28]. In our study, γδ TCR deficiency resulted in significant reduction of Hmgb1 and INF-γ expression in the allograft, suggesting that the attenuation of CAV observed in γδ TCR−/− recipient may be attributable to the modulation of Hmgb1 and INF-γ expression.

Recent clinical and experimental transplantation studies have showed that the involvement of IL-17 in allograft rejection. Increased IL-17 mRNA and protein levels were observed in patients with transplant rejection [29, 30]. In our previous experiment, NKG2D blockade significantly decreased IL-17-producing γδ T cells infiltration in cardiac allograft during acute rejection [8]. Now, in our current study, we showed that, during chronic cardiac transplant rejection, both gene and protein levels of IL-17 were significantly decreased in γδ TCR−/− recipient. This was consistent with previous study suggesting that IL-17 contributes to the development of chronic rejection in a murine heart transplant model [31, 32].

Regulatory T cells are known as a critical factor in expansion and activation of effecter T cells, which will in turn determine the fate of the transplanted organ. There is a large body of evidence that a population of Foxp3+ regulatory T cells is induced or expanded in many experimental models of transplantation tolerance [33]. Active suppression by regulatory T cells has been found to be one of the important mechanisms for induction and maintenance of self-tolerance and unresponsiveness to allograft [34, 35]. Surviving allograft in γδ TCR−/− recipient was infiltrated with a significant number of CD4+CD25+Foxp3+ T cells and depletion of CD25+ cells resulted in early rejection of allograft in our findings suggested that the induction of regulatory T cells may serve to the beneficial effect of γδ TCR deficiency.

In conclusion, our results was the first time providing strong evidence that γδ TCR deficiency resulted in attenuated CAV and prolonged graft survival in murine models of cardiac transplantation. This effect coincides with low expression of Hmgb1, IFN-γ and IL-17 while increased number of CD4+CD25+Foxp3+ regulatory T cells, and depletion of regulatory T cells abrogated the prolonged allograft survival induced by γδ TCR deficiency. Given result from Itoh et al. indicating that IL-17 could suppress regulatory T cell expansion, we speculated that γδ TCR deficiency may promote regulatory T cell expansion through IL-17 inhibition. In addition, previous studies have indicated that γδ T cells participated in renal or liver transplant rejection in human, and the role of γδ T cells in human cardiac transplantation was not investigated now [4, 36]. Although further investigations are needed to fully clarify the precise molecular and cellular mechanism involved in the immunoregulation, the administration of γδ TCR blocker may be of therapeutic benefit in inducing long-term allograft survival.

Acknowledgment

This work was supported in part by National Natural Science Found of China Grants 81202335.

Disclosures

The authors have no conflict of interest to declare.

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