Graft-versus-host Disease

Increased Th17/Treg ratio in chronic liver GVHD

Abstract

The contribution of Th17 cells to chronic GVHD (cGVHD) has been demonstrated in cGVHD mouse models. However, their contribution to human liver cGVHD remains unclear. We evaluated Th17 cells in biopsies from a cohort of 17 patients with liver cGVHD. We observed a significant increase in Th17 cells in the liver of patients with cGVHD, as demonstrated by an increase in CCR6+, CD161+ and RORγt+ T cells (P=0.03, P=0.0001 and P=0.03, respectively). We also assessed the presence of Th1 and regulatory (Treg) T cells: the numbers of Th1 and Treg cells were very low, with no difference between the two groups (P=0.88 and P=0.12, respectively). Furthermore, Th17/Th1 and Th17/Treg ratios were significantly increased in the liver of patients with liver cGVHD (P=0.005 and P=0.002, respectively). This study provides evidence for an infiltration by Th17 cells in the liver of patients with cGVHD and an increased Th17/Treg ratio, suggesting a defect in the regulatory mechanism driven by Treg cells or an inappropriate activation of effectors cells, especially Th17 cells, or both mechanisms, in human liver cGVHD.

Introduction

Chronic GVHD (cGVHD) is the main cause of late non-relapse mortality (NRM) and morbidity after allo-SCT. In contrast to acute GVHD (aGVHD), the pathophysiology of cGVHD remains poorly understood. Whereas aGVHD is classically restricted to the skin, liver and gastrointestinal tract, cGVHD is often more extensive and can also affect the eyes, lung or mucosa.1 cGVHD presents clinical features that mimic autoimmune diseases such as systemic sclerosis, lupus erythematosus or Sjögren syndrome, and autoimmune-like manifestations are a defining characteristic of cGVHD.2 The well-established role of Th17 cells in the pathophysiology of autoimmune diseases3, 4, 5 raises the question about the role of Th17 cells in cGVHD. The contribution of Th17 cells to cGVHD has been investigated in chronic GVHD mouse models. Although cGVHD can occur in the absence of IL-17,6 most studies have suggested its pathogenic role.7, 8, 9 It can be noted that, in a mouse model of cGVHD, a significant amount of Th17 cell infiltrate was found in the liver of allogeneic recipients as compared with syngeneic recipients.10 Furthermore, one of these studies indicated that the expansion of Th1 and Th17 cells is favored by the progressive loss of CD4+CD25+Foxp3+ regulatory T cells (Treg), leading to cGVHD onset.8 In humans, only a few studies explored the contribution of Th17 cells to cGVHD pathogenesis, and most studies have focused on skin cGVHD. Ritchie et al.11 showed that Th17-related cytokines are increased in patients with cGVHD. Furthermore, Dander et al.12 found a significant increase in circulating Th17 cells in patients with active cGVHD. With this background, the current study aimed to investigate the balance of Th17, Th1 and Treg cells in the liver of patients diagnosed with cGVHD.

Patients and methods

Study design

Seventeen patients who underwent allo-SCT and who developed liver cGVHD at the University-Hospital of Nantes (Nantes, France), and for whom biopsies were available, were included in this study. Liver cGVHD was proven histologically from a liver biopsy performed at the time of the first hepatic symptoms declaration or during their reappearance and prior to initiation or resumption of corticosteroid treatment. Hepatic cGVHD diagnosis was retained after exclusion of other differential diagnoses by physical examination and standard screening tests. Patients with new drug exposure or recent change in the dose schedule were excluded. Routine bacterial and fungal cultures were performed to exclude ongoing infection, and viral hepatitis caused by hepatitis A, B and C, VZV, HSV, adenovirus (ADV) and CMV was ruled out by serologic testing or PCR analysis. The control group included eight patients (three male and five female patients) without hematological malignancies. These control liver biopsies were performed for different indications (sleeve gastrectomy for morbid obesity, n=2, and adjacent tumor surgery, n=6). The median age was not significantly different compared with the cGVHD group (47 (range, 31–80) years vs 54 (27–71 years; P=0.70). All patients from the control group presented a histologically proven normal liver biopsy. All patients were enrolled in clinical research protocols approved by local ethical committees. Written informed consent was obtained in accordance with the principles of the declaration of Helsinki. Patients, donors and allo-SCT characteristics of the cGVHD group are summarized in Table 1.

Table 1 Patients, disease and transplant characteristics

Morphological studies

One liver biopsy was analyzed for each patient. Liver biopsies were performed according to standard procedures. In the cGVHD study group, the median time of biopsies was 264 (range, 119–1121) days after allo-SCT and 6.5 (range, 0–243) days after the first diagnosis of cGVHD. Liver cGVHD diagnosis was confirmed histologically on hematoxylin and eosin-stained (HES) sections according to the current recommendations.13 The liver biopsies from the eight patients of the control group were also examined on (HES) sections. In addition, none of the patients from the control or study group had evidence of viral infection.

Immunohistochemical studies

Immunohistochemical analysis was performed on 5 μm formalin-fixed, paraffin-embedded sections using an indirect immunoperoxidase method. Slides were stained with the following primary Abs: CD8 (cloneC8/144B; DakoCytomation, Glostrup, Denmark), granzyme B (clone GrB-7; DakoCytomation) and Foxp3 (clone 236A/E7; Abcam, Paris, France) for Treg cells; Tbet (clone 4B10; Santa Cruz Biotechnology, Santa Cruz, CA, USA) for Th1 cells; and CD161 (mouse MoAb, clone B199.2; AbDSerotec, Colmar, France), CCR6 (clone 53103; R&D Systems, Abingdon, UK) and RORγt (rabbit polyclonal Abs; Abcam, Paris, France) for Th17 cells. The immunological reaction was visualized with the bond polymer refine detection system and 3,3-diaminobenzidine tetrahydrochloride as the chromogen. In negative control experiments the primary Abs were omitted. A quantitative evaluation of the expression of the Ags was performed in a blinded manner independently by two examiners (FM and CB) by counting the number of positive cells in the whole biopsy at × 200 magnification (mean 10 fields) for each sample.

Statistical analysis

Clinical and morphological data, as well as the numbers of CD8-, granzyme B (GrB)-, Tbet-, Foxp3-, CD161-, CCR6- and RORγt-positive cells, were entered into a database. The Mann–Whitney non-parametric test was used to compare patient age and to assess the correlation between the number of CD8, GrB, Th1, Th17 and Treg cells and the presence of liver cGVHD. For patient gender, comparison between the cGVHD group and the control group was carried out using the chi-square test. All data were computed using GraphPad Software (GraphPad Prism, San Diego, CA, USA).

Results

Activated cytotoxic CD8+ and Gr B +cells infiltrate the liver of patients with liver cGVHD

The histological diagnosis of liver cGVHD was first confirmed. All HES sections of liver biopsies were retrospectively and blindly reviewed. All patients in the control group presented a histologically normal liver biopsy, whereas all patients in the cGVHD group presented with histological damage compatible with liver cGVHD, mainly bile duct damages with mild lymphocytic infiltration in the portal space and in the bile duct epithelium going up to the destruction of bile ducts in some cases (Figure 1a). Then, we sought to assess the presence of CD8+ T cells in the liver of cGVHD patients. We found that the number of CD8+ T cells was significantly increased in the liver of patients with liver cGVHD compared with the control group (P=0.02) (Figure 1b). We observed a predominantly portal infiltration of CD8+ cells in close contact with epithelial cells of the bile duct and associated in some cases with bile duct destruction (Figure 1a). We next assessed whether those CD8+ cells corresponded to activated cytotoxic T cells using GrB immunostaining. We found that the number of GrB+ cells paralleled the number of CD8+ T cells and was significantly increased in the liver of patients with liver cGVHD (P=0.0002) (Figures 1a and c).

Figure 1
figure1

CD8- and GrB-positive T cells infiltrate the liver of patients with liver cGVHD. Formalin-fixed and paraffin-embedded tissue samples of liver biopsies. A total of 25 biopsies were analyzed morphologically and by immunohistochemistry: 17 had liver cGVHD and 8 control patients did not. (a) Example of an HES section of liver biopsy showing bile duct damage up to destruction in cGVHD (lower panel) but not in healthy donors (upper level) (original magnification × 400). For quantitative evaluation, the mean number of positive cells for each marker was retained for the statistical analysis. (a) CD8- and GrB-positive mononuclear cells are significantly increased in the liver of patients with liver cGVHD (lower panel) compared with healthy donors (upper panel) (original magnification × 400). (b,c) Quantitative evaluation of CD8 (b) and GrB (c)-positive cells are represented. The mean values and s.e.m. for each group are represented.

Th17 cells infiltrate the liver of patients with liver cGVHD

Previous mouse studies suggested that Th17 cells are critical for injury in cGVHD. Thus, we hypothesized that Th17 cells could preferentially accumulate within the target tissues in human cGVHD. For the purpose of identification of Th17 cells we used the mucosal chemokine receptor CCR614, 15 and the C-type lectin-like receptor CD161, both of which are well-established markers of human Th17 cells.16 Using these two markers, we observed that the absolute number of CD161+ and CCR6+ cells was significantly higher in the liver of patients with cGVHD compared with the liver of patients in the control group (P=0.0001 and P=0.03, respectively; Figures 2a–c). These cells were mainly observed in the portal space, paralleling the localization of the CD8+ and GrB+ cells. In order to further confirm the Th17 cell infiltration, we also assessed the expression of RORγt, the key transcription factor that orchestrates the differentiation of Th17 cells.17 In keeping with the increase of CCR6+ and CD161+ T cells, RORγt+ cells (with nuclear staining) were significantly increased in the liver of patients with cGVHD compared with the liver of patients of the control group (P=0.03) (Figures 2a and d).

Figure 2
figure2

Th17 cells infiltrate the liver of hepatic cGVHD patients. (a) CCR6-, CD161- and RORγt-positive mononuclear cells are significantly increased in the dermis of liver with hepatic cGVHD (lower panel) compared with healthy donors (upper panel) (original magnification × 400). (bd) Quantitative evaluation of CCR6 (b), CD161 (c) and RORγt (d)-positive cells are represented. The mean values and s.e.m. for each group are represented.

Th17/Th1 and Th17/Treg ratios are increased in the liver of patients with liver cGVHD

We also assessed the presence of T cells expressing Tbet (the transcription factor characterizing Th1 cells) and Foxp3 (the master regulator gene of Treg cells) in the liver biopsies of the two groups of patients. Interestingly, there was no difference in the number of Th1 and Treg cells between the two groups (P=0.88 and P=0.12, respectively). These cells were scarce, observed both in the portal space (never in close contact with the epithelial cells of bile duct) and in the lobule inside sinusoids (Figures 3a–c). Finally, we looked at Th17/Th1 and Th17/Treg ratios in liver cGVHD. Considering that RORγt is the key transcription factor of Th17 cells and the more specific hallmark of Th17 cells, we defined the Th17/Th1 and Th17/Treg ratios as the ratio of RORγt-positive cells/Tbet-positive cells and that of RORγt-positive cells/Foxp3-positive cells, respectively. Both Th17/Th1 and Th17/Treg ratios were significantly increased in the liver of patients with liver cGVHD (P=0.005 and P=0.002, respectively; Figures 3d and e), suggesting an impaired regulatory response in the liver of patients with cGVHD.

Figure 3
figure3

Th17/Th1 and Th17/Treg ratios are increased in hepatic cGVHD. (a) The number of Tbet- and Foxp3-positive mononuclear cells in the liver of patients with liver cGVHD (lower panel) and in healthy donors (upper panel) was very low, and was identical in both groups (original magnification × 400). (b, c) Quantitative evaluation of CD8 (b) and GrB (c)-positive cells is represented. The mean values and s.e.m. for each group are represented. (d, e) Th17/Th1 (d) and Th17/Treg (e) ratios are increased in the liver of patients with liver cGVHD compared with healthy donors.

Discussion

The current study sheds some light on the role of Th17 and Treg cells in the context of liver cGVHD. Using well-established specific markers, we showed that (i) the number of activated cytotoxic CD8+ and GrB+ T cells was significantly increased in liver cGVHD, compared with the control group of patients; (ii) the number of Th17 cells was significantly increased in liver cGVHD compared with the control group of patients; and (iii) the number of Th1 and Treg cells was very low, not statistically different between the two groups of patients. Although immune dysfunction and autoreactivity may contribute to cGVHD pathophysiology, our study suggests a role for cytotoxic CD8+ donor T cells in cGVHD, especially in the liver. Only scarce information is available on the role of CD8+ T cells in cGVHD. Grogan et al.18 showed an expansion of activated CD8+ effector T cells in the blood of patients with cGVHD, the CD8+ central memory T cells being increased in cGVHD as well.19 In contrast, D’Asaro et al.20 observed a decrease in circulating CD8+ naive T cells in cGVHD patients; however, in this study all patients were receiving immunosuppressive treatment at the time of analysis. On the other hand, our study provides evidence that liver cGVHD is associated with a liver infiltration of CD8+ T cells, especially in the portal space, in close contact with epithelial cells and associated with bile duct damage; furthermore, these cells are activated, as evidenced by GrB expression, the protein associated with cytotoxic granules. Thus, these data strongly suggest that CD8+ T cells are part of the effector cells involved in liver cGVHD and likely contribute to tissue damage.

Croudace et al.21 have recently shown a decrease in CD4+ T cells expressing CXCR3 in the blood of patients with active cGVHD, whereas CD4+ T cells were increased in skin biopsies of the same patients. Furthermore, the CXCR3-binding chemokines, CXCL9, CXCL10 and CXCL11, were significantly increased in the serum of patients with cutaneous cGVHD. These data established a role for CD4+ T cells in cGVHD. However, controversies remained regarding the role and nature of the different CD4+ T-cell subsets. Although Th2 immune responses have long been considered responsible for the development of cGVHD,22 several studies have recently explored the role of Th1 and Th17 cells mainly in cGVHD mouse models, concluding on their pathological role.8, 10 However, analysis on their role in humans is scarcer. Some studies observed an increase in cytokines related to Th1 and Th17 in the blood of patients with cGVHD.11, 23, 24 Furthermore, Dander et al.12 observed a significant increase in the number of Th17 cells in the blood of patients with active cGVHD, and a high number of RORγt+ cells infiltrating the skin and liver of patients with cGVHD compared with healthy donors. Unfortunately, because of the small sample size, the latter findings in the skin and the liver were not statistically significant. Thus, our findings confirmed and extended the knowledge about the pathogenic role of Th17 cells in liver cGVHD. Furthermore, the increased Th17/Th1 ratio in cGVHD highlights the predominant role of Th17 cells over Th1 cells in liver cGVHD pathogenesis. Also, it is worth mentioning that these results are in line with the increased IFN-γ production in the blood of patients with cGVHD.25 Indeed, Th17 and Th1 cells present a close relationship and plasticity,26 and some RORγt+ Th17 cells can produce both IFNγ and IL-17A especially in an inflammatory setting, when the precursors of Th17 cells are in the dominant presence of IL-23 and not of TGF-β.27 Furthermore, if Th1 cells are not observed in active liver cGVHD at the time of the biopsy, we can speculate that they act earlier in the initial stages of the disease.

Regarding Treg cells, published data are more homogeneous. Most studies showed a decreased frequency of Treg in the blood of patients with cGVHD28, 29 Furthermore, our results in liver cGVHD are in accordance with those of Dander et al.12 in skin and liver cGVHD: the number of Foxp3+Treg cells was very low and comparable in cGVHD liver biopsies and in healthy donor liver biopsies. Furthermore, our results suggest that there is an imbalance in liver cGVHD between Treg and Th17 cells as we found an increased Th17/Treg ratio. In contrast, Ratajczak et al.30 found a decreased Th17/Treg ratio in cutaneous cGVHD. However, they did not analyze active cGVHD but chronic lichenoid skin lesions, and the number of patients included was lower than in our study (only five patients with cGVHD). This imbalance in the Th17/Treg ratio in liver cGVHD suggests the existence of a defect in the regulatory mechanisms driven by Treg cells or an inappropriate activation of effector cells, especially Th17 cells, or both mechanisms. One must acknowledge that one potential weakness of this study is the comparison of cGVHD patients with healthy controls. The ideal control would have been liver biopsies from patients who underwent allo-SCT and who did not develop any sign of cGVHD, which was impossible for obvious ethical reasons.

In conclusion, our study provides a framework for a regulatory defect and role of Th17-mediated response in human liver cGVHD, raising the prospect of future innovative approaches to optimize immunosuppression regimens for the treatment of cGVHD by targeting the Th17 response. There are already clinical trials aiming to block the Th17 response using anti–IL-17 MoAbs for autoimmune diseases.31, 32

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Acknowledgements

The authors acknowledge the technical and logistical support of S Blandin, C Deleine, S Leclercq and V Dehame. We also thank the nursing staff for providing excellent care for our patients, and the following physicians: N Blin, A Clavert, V Dubruille, T Gastinne, JL Harousseau, S Le Gouill, B Mahe, F Mechinaud and F Rialland for their dedicated patient care. FM and EB were supported by educational grants from the ‘Association for Training, Education and Research in Hematology, Immunology and Transplantation’ (ATERHIT). This work was supported by funding as part of the CESTI project (Nantes, France). We also thank the ‘Région Pays de Loire’, the ‘Association pour la Recherche sur le Cancer (ARC; grant #3175 to MM and BG)’, the ‘Fondation de France’, the ‘Fondation contre la Leucémie’, the ‘Agence de Biomédecine’, the ‘Association CentpourSang la Vie’, the ‘Association Laurette Fuguain’, the IRGHET and the ‘Ligue Contre le Cancer’ (Comités Grand-Ouest) for their generous and continuous support of our clinical and basic research work. Our transplant programs are supported by several grants from the French national cancer institute (PHRC, INCa to MM). The authors acknowledge the continuous support of the cell banking facility (‘tumorotheque’) of the CHU de Nantes.

Author contributions

All authors listed in the manuscript have contributed substantially to this work: conception and design: Florent Malard, Mohamad Mohty, Béatrice Gaugler, Céline Bossard; financial support: Mohamad Mohty, Marc Grégoire; administrative and logistical support: Jean-François Mosnier, Mohamad Mohty, Béatrice Gaugler, Marc Grégoire; provision of study materials and patients care: Eolia Brissot, Patrice Chevallier, Thierry Guillaume, Jacques Delaunay, Philippe Moreau, Mohamad Mohty; experimental work: Florent Malard, Céline Bossard; collection and assembly of clinical data: Florent Malard, Eolia Brissot, Mohamad Mohty; data analysis and interpretation: Florent Malard, Céline Bossard, Mohamad Mohty, Béatrice Gaugler; manuscript writing: Florent Malard, Mohamad Mohty, Céline Bossard, Béatrice Gaugler; final approval of manuscript: all co-authors.

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Malard, F., Bossard, C., Brissot, E. et al. Increased Th17/Treg ratio in chronic liver GVHD. Bone Marrow Transplant 49, 539–544 (2014). https://doi.org/10.1038/bmt.2013.215

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Keywords

  • Allo-SCT
  • liver cGVHD
  • Th17 cells
  • regulatory T cells

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