A previous paper has reported that blockade of NKG2D was effective in protecting allograft in murine models of cardiac transplantation, but the mechanism of NKG2D blockade on attenuated cardiac allograft vasculopathy (CAV) was still unknown. In our current study, we found that wild-type recipients treated with anti-NKG2D monoclonal antibody (mAb) plus cytotoxic T lymphocyte antigen (CTLA)-4-immunoglobulin (I)g showed prolonged allograft survivals (>90 days, P < 0·001) significantly and attenuated CAV. These in-vivo results correlated with reduced alloantibody production, low expression of interleukin (IL)-17 and IL-6, while infiltration of regulatory T cells increased. IL-6 administration induced shorter allograft survival and higher CAV grade in CTLA-4–Ig plus anti-NKG2D mAb-treated recipients, whereas IL-17 had no significant effect on allograft survival and CAV grade in CTLA-4–Ig plus anti-NKG2D mAb-treated recipients. Furthermore, the prolonged allograft survival induced by NKG2D blockade was abrogated partially with depletion of regulatory T cells. In conclusion, blockade of NKG2D combined with CTLA-4–Ig attenuated CAV and this effect was associated with lower alloantibody production, inhibited IL-6 expression and enhanced expansion of regulatory T cells.
Cardiac transplantation is the last resort for patients with end-stage heart failure. Short-term patient survival of acute rejection has been improved substantially during recent years but, so far, long-term survival has not been raised dramatically. The predominant obstacle has been cardiac allograft vasculopathy (CAV) . Hence, research effort has been directed at exploring strategies that can overcome the shortcoming of conventional immunosuppressives to inhibit the development of CAV effectively.
The receptor-ligand pair B7–CD28 was essential for the initiation and amplification of the T cell-dependent immune response. B7–CD28 interactions provided second signals necessary for optimal T cell activation and interleukin (IL)-2 production. Blockade of B7–CD28 has been reported to prolong allograft survival in cardiac transplantation due to its function in inhibiting T helper type 1 (Th1) cytokine expression [2-5]. NKG2D is an activating receptor expressed on all natural killer (NK) T cells, a subset of γδ T, activated CD8+ T cells and a subset of CD4+ T cells [6-8]. Recent papers have indicated that NK cells play a necessary role in the development of chronic rejection, and depletion of NK cells prevents CAV [9, 10]. T cells, including CD4+ T cells, CD8+ T cells and γδ T cells, are also involved in the development of CAV [11, 12]. NKG2D and its ligands, including major histocompatibility complex (MHC) I chain-related genes MICA/B, retinoic acid early inducible (Rae-1) and minor histocompatibility antigen H60 molecules, play an important role in the linkage between innate and adpative immunity [13, 14]. Adaptor molecules DAP10 and DAP12 binding to NKG2D are thought to have a particularly important role in transplant immunity .
A previous paper has indicated that the addition of anti-NKG2D monoclonal antibody (mAb) treatment to CTLA-4–Ig extends cardiac allograft survival, but the mechanism is unknown . A recent paper from our colleague also showed that blockade of NKG2D synergized with CTLA-4–Ig in alleviating acute rejection in cardiac transplantation, and this effect coincided with high expression of IL-4 as well as an increased number of alternatively activated macrophages (AAMs), and low expression of interferon (IFN)-γ and a reduced number of IL-17-producing γδ T cells infiltration . However, to our knowledge, the role of NKG2D on CAV has never been investigated.
In our current study, we investigated the impact of NKG2D on chronic transplant rejection and found that NKG2D blockade attenuated CAV and this was associated with reduced alloantibody production, inhibited IL-6 expression and enhanced expansion of regulatory T cells.
Materials and methods
Adult male BALB/c (H-2d) mice were obtained from the Center of Experimental Animals, Tongji Medical College of Huazhong Science and Technology University, China. C57BL/6 (H-2b) mice were from the Institute of Laboratory Animal Sciences of the Chinese Academy of Medical Sciences (Beijing, China). B6.C-H-2bm12KhEg (H-2bm12) mice were purchased from the Jackson Laboratory (Bar Harbor, ME, USA). All the mice were 8-week-old males, 25–30 g in weight, housed in a specific pathogen-free facility with regular food and water ad libitum. Experiments were approved by the Institutional Animal Care and Use Committee at Tongji Medical College (Wuhan, China).
Reagents and antibodies
Anti-mouse NKG2D mAb (HMG2D) was purchased from BioXCell (West Lebanon, NH, USA). The antibody was confirmed to have comparable activity to another clone of anti-NKG2D mAb, CX5 [8, 18]. Anti-mouse mAbs to CD4, CD8, CD68, IL-17, forkhead box protein 3 (FoxP3) and C4d were from eBioscience (San Diego, CA, USA). Recombinant monoclonal rat anti-mouse IL-17 and IL-6 were from R&D Systems (Minneapolis, MN, USA). CTLA-4–Ig fusion protein was obtained from Bristol-Myers Squibb (New York, NY, USA).
Heterotopic cardiac transplant and post-transplant therapies
BALB/c (H-2d) hearts were transplanted heterotopically into C57BL/6 (H-2b) recipient mice as allograft and comprised the control group . Hearts of B6.C-H-2bm12KhEg (H-2bm12) were transplanted heterotopically into MHC class II-mismatched C57BL/6 (H-2b) mice, which is an established murine model of chronic allograft rejection without immunosuppression. The mice were anaesthetized by a single intraperitoneal injection of ketamine/xylazine (100:10 μg/kg). The strength and quality of cardiac impulses were graded by palpation on a daily basis. In the CTLA-4–Ig-treated group, the recipients were administered 200 μg CTLA-4–Ig on days 0, 3, 7, 10 and 20. The anti-NKG2D mAb-only recipients (n = 6) were treated with 250 μg anti-NKG2D mAb (HMG2D) twice weekly for 6 weeks. In the combined therapy group, recipients (n = 18) were treated by intraperitoneal injection with 200 μg CTLA-4–Ig on days 0, 3, 7, 10 and 20 in combination with 250 μg anti-NKG2D mAb (HMG2D) twice weekly for 6 weeks . In the combined therapy group, six animals were killed on days 60 and 90 post-transplant, respectively. In the CTLA-4–Ig-only group, cardiac allografts were obtained when rejection occurred. For regulatory T cell depletion, recipients were injected intraperitoneally with 500 μg of anti-mouse CD25 mAb (PC61, Bioexpress cell culture; Bioexpress, Kaysville, UT, USA) 1 day before transplantation . Mouse IL-17 or IL-6 was administered twice weekly with a 3 μg/injection dose until rejection . In the B6.H-2bm12→B6.H-2b group (n = 6), cardiac allografts were obtained when rejection occurred. In the B6.H-2bm12→B6.H-2b + anti-NKG2D group (n = 12), six animals were killed on day 58 post-transplantation.
Histopathology and immunohistochemistry
Cardiac allograft tissues were stained with haematoxylin and eosin (H&E) and elastic-van Garson's (EvG) stains . 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, Atlanta, GA, USA); the luminal occlusion rate was calculated using 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 described recently in detail . Immunohistochemical staining for C4d, IL-17 and FoxP3 was also carried out as described previously . To evaluate cell infiltration, five fields were selected randomly from one section to count the number of positive cells in each field.
Total RNA was isolated from allografts using a Mini Kit (Qiagen, Hilden, Germany). Contaminating genomic DNA was removed by digestion with Rnase-free Dnase (Qiagen). The integrity of isolated RNA was verified by analytical agarose gel electrophoresis. Two micrograms of Dnase-treated RNA was used to synthesize the first strand of the cDNA synthesis kit. The primers and for IL-17 and IL-6 in were used as described previously [16, 25].
Serum was collected from recipients on day 60 post-transplantation by retro-orbital bleeds and analysed using the Bioplex System (Bio-Rad Laboratories, Hercules, CA, USA), according to the manufacturer's instructions. The concentration of IL-6 in serum was measured using the multiplex cytokine Bio-Rad assay system (Bio-Rad) .
According to a previous study reported by Gorbacheva et al., infiltrated cells in the allograft were isolated . Cells were stimulated with 0·1 μg/ml phorbol myristate acetate (PMA) (Sigma, St Louis, MO, USA), 1 μg/ml ionomycin (Sigma) and brefeldin A (10 μL/6 ml; Biosciences) for 4 h at 37°C. 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 Biosciences, San Jose, CA, USA). Isotype controls were given to enable the correct compensation and to confirm antibody specificity.
Western blot analysis
Tissue homogenization, electrophoresis and protein transference were performed as described elsewhere . Membranes were processed for immunodetection using rabbit anti-cat polyclonal antibody, sheep anti-Cu/Zn-superoxide dismutase (SOD) polyclonal antibody, goat anti-Prx-6 polyclonal antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA), rabbit anti-IL-6 polyclonal antibody and mouse anti-tumour necrosis factor (TNF)-α monoclonal antibody (Abcam Inc., CA, Burlingame, USA) as primary antibodies. The bound primary antibodies were detected using rabbit anti-sheep, rat anti-mouse or goat anti-rabbit horseradish peroxidase-conjugated secondary antibodies and membranes were revealed for chemiluminescence. The resultant autoradiographs were analysed quantitatively with an image densitometer (Image Master VDS CI; Amersham Biosciences Europe, Italy). The molecular weights of the bands were determined by reference to a standard molecular weight marker (RPN 800 rainbow full range; Bio-Rad). Results from each membrane were normalized using the Ponceau Red method.
Serum alloantibody was measured by flow cytometry using frozen donor splenocytes, as reported previously . Briefly, 5 × 105 donor splenocytes were incubated with 50 μl of recipient serum at 4°C for 30 min. After washing, cells were incubated with fluorescein isothiocyanate (FITC)-anti-mouse IgM or anti-mouse IgG (BD Pharmingen, CA, USA) at 4°C for 30 min, washed twice with a flow cytometry buffer and analysed using a FACScalibur (BD Biosciences). Results are expressed as the mean fluorescence intensity (MFI) of the post-transplant minus pretransplant values. The response to autologous splenocytes served as negative controls.
Data are expressed as mean ± standard error of the mean. Kaplan–Meier methods were used to calculate the graft survival. 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.
NKG2D blockade prolonged allograft survival and attenuated CAV grade
We first examined the impact of additional NKG2D blockade to CTLA-4–Ig therapy on heart allograft survival. After cardiac transplantation, animals were treated with CTLA-4–Ig alone or in combination with anti-NKG2D mAb. Control recipients with no treatment or recipients treated with anti-NKG2D mAb alone acutely rejected heart allografts (MST = 7·5 days and 8·5 days, respectively). Treatment with CTLA-4–Ig alone resulted in a modest prolongation of allograft survival (MST = 60 days). In contrast, allografts of recipients treated with CTLA-4–Ig plus anti-NKG2D mAb showed long-term allograft survival (MST > 90, P < 0·001 compared with other groups; Fig. 1a). On day 60 post-grafting, the vessels in cardiac allografts from the CTLA-4–Ig-only group were almost obstructed (mean CAV grade: 2·53 ± 0·13) and from the combined therapy group were slightly affected (mean CAV grade: 1·20 ± 0·11, P < 0·001) (Fig. 1b,c). On day 90 post-transplantation, the mean CAV grade in cardiac graft from combined therapy was 1·46 ± 0·13 (P > 0·05 compared with vessels on day 60 post-grafting from the combined therapy group). In order to investigate further the role of NKG2D on CAV, we established a chronic allograft rejection model with B6.C-H-2bm12KhEg (H-2bm12) hearts transplanted heterotopically into C57BL/6 (H-2b) mice. The results showed that NKG2D blockade prolonged cardiac allograft survival (MST = 79 days in B6.H-2bm12→B6.H-2b group + anti-NKG2D group, P < 0·05 compared with B6.H-2bm12→B6.H-2b group, MST = 58 days) and attenuated CAV grade on day 58 post-transplantation (mean CAV grade: 1·25 ± 0·13, P < 0·05 compared with B6.H-2bm12→B6.H-2b group, mean CAV grade: 2·41 ± 0·15) (Fig. 1d,e).
NKG2D blockade attenuated leucocyte infiltration and alloantibody production in cardiac allograft
We examined the number of graft-infiltrating immune cells. On day 60 post-grafting, the number of graft-infiltrating CD4+ T, CD8+ T cells and CD68+ macrophages were markedly lower in recipients treated with anti-NKG2D mAb plus CTLA-4–Ig (Fig. 2a). The C4d deposition in allografts from anti-NKG2D mAb plus CTLA-4–Ig-treated recipients was much less compared with that from CTLA-4–Ig-only recipients (Fig. 2b). We also examined the impact of NKG2D blockade on alloantibody production in heart allograft recipients. The results showed that the levels of both anti-donor IgM and IgG were significantly lower with the addition of anti-NKG2D mAb (Fig. 2c).
Inhibited IL-6 expression contributed to attenuated CAV induced by NKG2D blockade
First, we examined IL-17 expression in allografts. The results showed that, with the additional use of anti-NKG2D mAb, there was much less IL-17+ cell infiltration and IL-17 mRNA expression. We also found that IL-17 was produced predominantly by γδ T cells rather than CD4+ T cells or CD8+ T cells infiltrated into the cardiac allografts. Furthermore, using anti-NKG2D mAb, the number of IL-17+ γδ T cells was decreased significantly on day 60 post-transplantation (Fig. 3a–c). We then detected the expression of IL-6 in allograft and peripheral blood, and found that IL-6 mRNA and protein levels in cardiac graft and serum IL-6 expression were much lower in recipients treated with additional NKG2D blockade (Fig. 3d–f).
Previous papers have shown that IL-6 is essential in the induction of Th17 cells in both humans and mice . IL-6 is a pleiotropic cytokine produced by multiple cell types that has well-described functions in both innate and adaptive immune responses . In order to investigate the role of IL-17 and IL-6 in ameliorated CAV induced by NKG2D blockade, we administered IL-17 or IL-6 into CTLA-4–Ig plus anti-NKG2D mAb-treated recipients. The results showed that IL-6 administration resulted in reduced allograft survival (MST = 78 days, P < 0·05 compared with the CTLA-4–Ig + anti-NKG2D mAb group) and increased CAV grade (CAV grade: 1·93 ± 0·15, P < 0·05 compared with the CTLA-4–Ig + anti-NKG2D mAb group), whereas IL-17 had no significant effect on allograft survival (MST = 88 days, P > 0·05 compared with the CTLA-4–Ig + anti-NKG2D mAb group) and CAV grade (CAV grade: 1·47 ± 0·13, P > 0·05 compared with the CTLA-4–Ig + anti-NKG2D mAb group) (Fig. 4).
Prolonged allograft survival is partially dependent upon enhanced regulatory T cell expansion
First, we investigated the expression of regulatory T cell in allograft and spleen; the results indicated that in both allograft and spleen, the proportion of CD25+FoxP3+ regulatory T cells in CD4+ T cells (allograft: 9·967 ± 0·917% versus 3·917 ± 0·450%, P < 0·005; spleen: 6·467 ± 0·905% versus 2·067 ± 0·360%, P < 0·005) and the number of FoxP3+ regulatory T cells were increased significantly in CTLA-4–Ig plus anti-NKG2D mAb-treated recipients compared with CTLA-4–Ig-only recipients (Fig. 5). We then studied the function of regulatory T cells in transplant rejection; we injected anti-CD25 mAb into allograft recipients treated with anti-NKG2D mAb plus CTLA-4–Ig, and found that the survival time was reduced significantly (P < 0·05 compared with the CTLA-4–Ig + anti-NKG2D mAb group, MST = 70 days) (Fig. 6).
Our current study demonstrated that the addition of anti-NKG2D mAb to CTLA-4–Ig therapy attenuated CAV, and this was associated with reduced alloantibody production, inhibited IL-6 expression and enhanced regulatory T cell expansion.
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 upon several factors, such as precursor frequency, factors involved in antigen presentation and co-stimulation and proinflammatory signals produced by the innate immune system . Cells of the macrophage lineage are a major component of the infiltrate in allografts undergoing T cell-mediated rejection . Macrophages are involved in innate and adaptive immunity during allograft rejection, playing a key role in the initiation and effector phases of the immune response [33, 34]. On the basis of previous findings, our experiment proved further that decreased numbers of effector T lymphocyte and macrophage infiltration may contribute to reducing CAV by NKG2D blockade.
Several mechanisms have been implicated in complement activation post-transplantation. Capillary deposition of the C4d complement fragment and alloantibody production were shown previously to identify humoral rejection in human heart and kidney transplants, and have been discussed repeatedly in the pathogenesis of CAV [35, 36]. Long-term activation of the complement system because of recurrent or prolonged antibody-mediated alloreactions may result eventually in the development of CAV . Furthermore, some research has indicated that macrophages were the principal cells involved in humoral rejection-related myocyte injury and were associated with C4d staining [38, 39]. Based on the above, and the reduced alloantibody production, decreased C4d deposits and CD68+ macrophage infiltrates in our experiment, we conclude that humoral rejection could be inhibited partially by NKG2D blockade.
Recent clinical and experimental transplantation studies have shown the involvement of IL-17 in allograft rejection. Itoh et al. showed that IL-17 contributes to the development of chronic rejection in a murine heart transplant model . IL-6 is a proinflammatory cytokine that plays a key role in immune responses, and recent studies have indicated that IL-6 is a key cytokine that determines the balance between FoxP3+ regulatory T cells and Th17 cells [41, 42]. In our current study we show that, during chronic cardiac transplant rejection, both gene and protein levels of IL-17 and IL-6 were decreased significantly with additional blockade of NKG2D. In our further study with IL-17 or IL-6 administration into CTLA-4–Ig plus anti-NKG2D mAb-treated recipients, we found that IL-6 expression resulted in reduced allograft survival time and increased CAV grade, whereas IL-17 did not alter allograft survival and CAV grade significantly. These data indicate that IL-6 might be a downstream target of IL-17 and inhibits IL-6 expression, contributing to attenuated CAV induced by NKG2D blockade.
Regulatory T cells are known as a critical factor in the expansion and activation of effector 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 are induced or expanded in many experimental models of transplantation tolerance . Active suppression by regulatory T cells has been found to be one of the important mechanisms for the induction and maintenance of self-tolerance and unresponsiveness to allografts [44, 45]. Surviving allografts treated with NKG2D blockade plus CTLA-4–Ig were infiltrated with a significant number of CD4+CD25+FoxP3+ T cells, and the depletion of CD25+ cells resulted in reduced allograft survival in our findings, suggesting that the induction of regulatory T cells might be a beneficial effect of NKG2D blockade. A previous paper by Roy et al. reported that NK cells activated by cytokines IL-12, IL-15 and IL-18 or by exposure to intracellular pathogens could reduce regulatory T cell expansion through NKG2D-dependent lysis . Combined with our study, we speculate that NKG2D blockade increases regulatory T cell expression through reducing NKG2D-dependent regulatory T cell lysis followed by attenuated transplant vasculopathy.
Prolonged administration of anti-NKG2D mAb might be a reason for the better effect in allograft survival compared with a report from Kim et al. ; this was consistent with the previous finding, that continued treatment with anti-NKG2D mAb was required to prevent autoimmune diabetes in the non-obese diabetic (NOD) mouse model . The results in our study demonstrate that anti-NKG2D mAb synergizes with CTLA-4–Ig in attenuating CAV in murine models of transplantation, and this effect is associated with reduced alloantibody production, inhibiting IL-6 expression and enhancing regulatory T cell expansion. Although further investigation is needed to clarify fully the precise molecular and cellular mechanism involved in immunoregulation, administration of the additional NKG2D blocker to CTLA-4–Ig could be of therapeutic benefit in attenuating CAV and inducing long-term allograft survival.