Graft-Versus-Host Disease

IL-4-producing CD8+ T cells may be an immunological hallmark of chronic GVHD

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Summary:

Chronic graft-versus-host disease (cGVHD) occurs in approximately 60–80% of those who survive over 100 days after allogeneic hematopoietic stem cell transplantation (allo-HSCT). However, the pathophysiology of cGVHD is poorly understood. To gain more insight into the immunological mechanism of cGVHD, we examine cytokine production of peripheral blood T cells from 19 patients in the chronic phase of allo-HSCT. The percentage of IFN-γ-producing CD8+ T cells among CD8+ T cells was significantly higher in patients with or without cGVHD than in normal control subjects (P<0.001). On the other hand, the percentage of IL-4-producing CD8+ T cells among CD8+ T cells was significantly higher in patients with cGVHD (mean 3.3%; range 1.3–8.2%) than in patients without cGVHD (mean 1.2%; range 0.8–1.7%) and normal control subjects (mean 1.1%; range 0.1–1.6%) (both P<0.001). By contrast, the percentage of IL-4-producing CD4+ T cells was not different among patients with and without cGVHD and normal controls. These findings suggest that IL-4-producing CD8+ T cells may be an immunological marker of cGVHD.

Main

Graft-versus-host disease (GVHD) is a major cause of morbidity and mortality after allogeneic hematopoietic stem cell transplantation (allo-HSCT),1 which is commonly classified into acute or chronic GVHD Acute GVHD (aGVHD) mainly develops in the skin, gut, and liver and directly contributes to the higher mortality rate in the early phase after allo-HSCT.2 On the other hand, chronic GVHD (cGVHD) usually develops in systemic organs and decreases the quality of life in the late phase after allo-HSCT.3, 4, 5, 6 Furthermore, elevated mortality is associated with the complications following cGVHD such as interstitial pneumonia and infections.7

GVHD is a complicated post transplant condition, and a myriad of factors are suggested to be involved in this process. The essential basis of GVHD is recognized to be primarily the immune responses that are evoked by the existence of allogeneic disparities between donor and recipient and that subsequently attack the recipient organs.1 The first process might be allo-antigen presentation to the donor T cells by antigen-presenting cells (APCs), especially dendritic cells. This interaction between APCs and T cells in microenvironments such as cytokines dictate the development of type 1 helper T-cells (Th1) or type 2 helper T-cells (Th2).8, 9 It is well established that Th1 cells produce cytokines such as IL-1, IL-2 and IFN-γ, while Th2 cells produce cytokines such as IL-4 and IL-10.

Accumulating evidence has highlighted the importance of Th1 balance in aGVHD.10, 11, 12, 13, 14, 15, 16, 17, 18, 19 However, the pathophysiology of cGVHD is much less well understood than that of aGVHD, and the current understanding of cGVHD is largely the result of murine studies. In the murine system, it has been shown that post-thymic CD4+ T cells are important in inducing cGVHD.20 In addition, several reports have demonstrated that type 2 cytokines are associated with cGVHD,21, 22 whereas other studies favor the significance of Th1 balance in cGVHD.23, 24, 25, 26

In this study, we analyzed the cytokine production of T cells in the chronic phase of patients who underwent allo-HSCT and found that IL-4-producing CD8+ T cells are closely associated with cGVHD.

Patients and methods

Patients

A total of 19 patients who underwent allo-HSCT between June 1994 and March 2001 and were alive over 100 days after allo-HSCT without relapse were enrolled in this study, for which informed consent was obtained from all patients. The patient characteristics are shown in Table 1. The group of patients with cGVHD showed some sign of cGVHD, while the group of patients without cGVHD did not show any sign of cGVHD at the time of examination. Of them 10 patients (six men and four women, mean age 33 years, range 20–47 years) developed cGVHD, while the remaining nine patients (six men and three women, mean age 29 years, range 13–39 years) did not show any clinical signs of cGVHD. The patients with or without cGVHD were examined at the median time of 1204 days (range 522–2840 days) and 1153 days (range 571–2798 days) after allo-HSCT, respectively. Five patients with cGVHD had acute myelogenous leukemia (AML), two had chronic myelogenous leukemia (CML), two had acute lymphoblastic leukemia (ALL) and one had severe aplastic anemia. Six patients without cGVHD had AML, one had CML and two had ALL. Eight patients with cGVHD received bone marrow transplantation (BMT) from their siblings, one patient received unrelated BMT and one patient received peripheral blood stem cell transplantation (PBSCT) from their siblings. On the other hand, seven patients without cGVHD received related BMT, one patient received unrelated BMT and one patient received related PBSCT. One patient with cGVHD and one patient without cGVHD received allo-HSCT from human leukocyte antigen (HLA) mismatched donors (both one locus; HLA-DR), while the remaining patients received HLA-full-matched grafts. As a conditioning regimen, seven patients with cGVHD received total body irradiation (TBI total 12 Gy), cyclophosphamide (CY) and cyclosporin A (CyA), two patients received TBI and CY and the remaining one patient was administered combinations of high-dose chemotherapy; busulfan (BU) and CY. On the other hand, six patients without cGVHD received TBI, CY and CyA, two patients received TBI and CY and one patient BU, CY and TBI. Three patients with cGVHD and two patients without cGVHD received a combination of tacrolimus (FK) and methotrexate (MTX) for prophylaxis of aGVHD, while others received combination of CyA and MTX. Five patients with cGVHD and four patients without cGVHD had previously developed grade II–IV aGVHD.27 Of 10 patients who had cGVHD at the time of examination, four with extensive cGVHD were being administered immunosuppressive agents or corticosteroids at the time of examination (one had received CyA, one had prednisolone (PSL) and two had a combination of PSL and FK). The cGVHD in these four patients was thus ameliorated and manifested as of the limited type (although their cGVHD is shown as ‘extensive’ in Table 1)28 at the time of examination. By contrast, the remaining six showed limited-type cGVHD and had not been administered any immunosuppressive agents or corticosteroids for at least 2 months before the examination. On the other hand, of nine patients who did not have cGVHD at the time of examination, three had limited type cGVHD prior to this study and had been successfully treated with immunosuppressive agents or corticosteroids (one had received FK and two had PSL), whereas the remaining six had not been received any immunosuppressive agents or corticosteroids for at least 2 months before the examination. All 19 patients were alive at the time of this analysis.

Table 1 Patient characteristics

Controls

For normal controls, we collected blood from 10 healthy adult volunteers (six men and four women, mean age 29 years, range 22–46 years). No patients or volunteers showed evidence of infection at the time of study.

Media

RPMI 1640 medium supplemented with 2 mM L-glutamine, 100 units/ml penicillin, 100 ng/ml streptomycin, and heat-inactivated 10% fetal bovine serum (Irvine Scientific, Santa Ana, CA, USA) was used throughout the experiments.

Count of absolute number of CD4+ or CD8+ T cells

The peripheral blood mononuclear cells (PBMCs) were freshly isolated by Lymphoprep (Nycomed Pharma, Oslo, Norway) gradient centrifugation of heparinized blood. The isolated PBMCs were stained with fluorescein isothiocyanate (FITC)-labeled CD4 (RPA-T4; Becton Dickinson, San Jose, CA, USA), phycoerythrin (PE)-labeled CD8 (HIT8a; Becton Dickinson) and phycoerythrin-cyanin 5.1 (PC5)-labeled CD3 (UCHT1; Beckman Coulter, Marseilles, France). These cells were washed with phosphate buffered saline (PBS) containing 0.2% bovine serum albumin, and the percentages of CD4+ or CD8+ T cells were then measured using a FACScan flow cytometer (Becton Dickinson). The absolute numbers of CD4+ or CD8+ T cells (/ml) were calculated by multiplication of the percentage of each cell-type with total PBMCs (/ml).

Detection of cytokines from T cells by intracellular staining

PBMCs were suspended in RPMI 1640 at a concentration of 3 × 106/ml. Cells were stimulated with 50 ng/ml phorbol-12-myristate-13-acetate (PMA) (Sigma, St Louise, MO, USA) and 1 μg/ml ionomycine (Sigma) in the presence of 2 μg/ml brefeldin A (ICN Biomedicals, Aurora, OH, USA) for 4 h at 37°C. The cells were then washed with PBS containing 0.2% bovine serum albumin and stained with PC5-labeled anti-CD4 (13B8.2; Beckman Coulter), or anti-CD8 (B9.11; Beckman Coulter) for 30 min in the dark. To evaluate the cytokine production, these cells were further manipulated using Fix & Perm cell Permeabilization Kit (CALTAG, Burlingame, CA, USA) and were stained with FITC-labeled anti-IFN-γ (4S.B3; Becton Dickinson) combined with PE-labeled anti-IL-4 (8D4-8; Becton Dickinson), or IL-10 (JES3-19F1; Becton Dickinson). These cells were washed with PBS and resuspended in 200 μl of 1% formaldehyde (in PBS, pH 7.2), and were then measured using a FACScan flow cytometer.

Detection of cytokine production from T cells incubated for 24 h

PBMCs were divided into CD4+ T cells and CD8+ T cells using anti-CD4-coated magnetic beads (Miltenyi Biotec, Bergisch Gladbach, Germany) and anti-CD8-coated magnetic beads (Miltenyi Biotec) among patients with cGVHD, patients without cGVHD and the normal control subjects. The >98% purity of these cells was proved by FACS analysis using anti-CD4 or anti-CD8 mAb. The isolated CD4+ and CD8+ T cells (1 × 106 cells) were cultured separately with 50 ng/ml PMA and 2 μg/ml ionomycine in 24-well flat-bottomed culture plates in 1000 μl of medium per well. After 24 h, the cytokines (IL-4, IL-10 and IFN-γ) in each supernatant were measured by ELISA using commercially available kits (Endogen, Woburn, MA, USA).

Statistical analysis

Continuous data were compared using Kruskal–Wallis test. When the difference was significant, we evaluated each two variables using Mann–Whitney U-test with the Stat View statistical program (Abacus Concepts, Berkeley, CA, USA). Differences were considered significant when tied P-values were less than 0.05.

Results

Absolute number of CD4+ T cells and CD8+ T cells

The absolute number of CD4+ T cells was not significantly different among normal controls, patients without cGVHD, and patients with cGVHD (control; mean 7.4 × 105/ml, range 5.3–11.9 × 105/ml, patients without cGVHD; mean 6.5 × 105/ml, range 3.4–9.7 × 105/ml, patients with cGVHD; mean5.9 × 105/ml, range 3.3–8.6 × 105/ml) (Figure 1a). Furthermore, the absolute number of CD8+ T cells was not significantly different among the three groups (normal controls; mean 4.4 × 105/ml, range 2.2–5.9 × 105/ml, patients without cGVHD; mean 6.5 × 105/ml, range 3.3–9.8 × 105/ml, patients with cGVHD; mean 6.3 × 105/ml, range 2.3–11.3 × 105/ml) (Figure 1b). In addition, the absolute number of CD4+ or CD8+ T cells in patients with cGVHD or without cGVHD was not significantly affected by the use of immunosuppressive agents or corticosteroids (Figure 2a, b).

Figure 1
figure1

The absolute counts of CD4+ and CD8+ T cells in PBMC. The open column is controls (n=10), the dotted column is patients without cGVHD (n=9), and the black column is patients with cGVHD (n=10). (a) The absolute number of CD4+ T cells. (b) The absolute number of CD8+ T cells.

Figure 2
figure2

The absolute counts of CD4+ and CD8+ T cells in PBMC. The open column is patients who were not receiving predonisolone or immunosuppressive agents. The shaded column is patients who were receiving predonisolone and/or immunosuppressive agents. (a) The absolute counts of CD4+ and CD8+ T cells of patients with cGVHD. Open column is n=6, shaded column is n=4. (b) The absolute counts of CD4+ and CD8+ T cells of patients without cGVHD. Open column is n=6, shaded column is n=3.

Increased percentage of IFN-γ-producing peripheral blood CD4+ T cells in two patient groups

We analyzed the cytokine production of the peripheral blood CD4+ T cells and CD8+ T cells using the intracellular cytokine staining method. The percentage of IL-4-producing CD4+ T cells among total CD4+ T cells was not significantly different among patients with cGVHD (mean 3.9%; range 2.4–7.7%), without cGVHD (mean 3.6%; range 0.7–7.6%) and normal control subjects (mean 2.8%; range 0.2–4.9%) (Figure 3a). On the other hand, the percentage of IFN-γ-producing CD4+ T cells was significantly higher in patients with cGVHD (mean 51.1%; range 18.6–80.7%) and without cGVHD (mean 37.7%; range 9.5–63.4%) than in the normal control subjects (mean 16.7%; range 6.7–38.9%) (P=0.0001, P=0.0073) (Figure 3b). However, the percentage of IFN-γ-producing CD4+ T cells was not statistically different between the two patient groups. To confirm the results from intracellular cytokine staining, we examined the titers of cytokines in the supernatants of 24-h-cultured CD4+ T cells by ELISA. Although the titer of IL-4 and IFN-γ from CD4+ T cells was not statistically different among patients with and without cGVHD and normal control subjects, the ELISA data were basically similar to that of intracellular cytokine staining (data not shown).

Figure 3
figure3

The percentage of IL-4 and IFN-γ -producing CD4+ T cells by intracellular cytokine staining. The open column is controls (n=10), the dotted column is patients without cGVHD (n=9), and the black column is patients with cGVHD (n=10). ***P<0.001. (a) The percentage of IL-4-producing CD4+ T cells among total CD4+ T cells. (b) The percentage of IFN-γ-producing CD4+ T cells among total CD4+ T cells.

Increased percentage of IL-4-producing peripheral blood CD8+ T cells in patients with cGVHD

The percentage of IL-4-producing CD8+ T cells was significantly higher in patients with cGVHD (mean 3.3%; range 1.3–8.2%) than in patients without cGVHD (mean 1.2%; range 0.8–1.7%) (P=0.0002) and normal control subjects (mean 1.1%; range 0.1–1.6%) (P=0.0006) (Figure 4a). There was no significant difference in the percentage of IL-4-producing CD8+ T cells between patients without cGVHD and normal control subjects. On the other hand, the percentage of IFN-γ-producing CD8+ T cells was significantly higher in patients with cGVHD (mean 74.5%; range 59.2–91.3%) and without cGVHD (mean 68.9%; range 34.1–93.7%) than in normal control subjects (mean 35.3%; range 20–58.4%) (P<0.0001, P=0.0007) (Figure 4b). However, it was not different between the two patient groups. Figure 5 shows representative data of intracellular cytokine staining of IL-4 and IFN-γ of CD8+ T cells. IL-4-producing CD8+ T cells were barely detected in the control subjects (Figure 5a) and the patients without cGVHD (Figure 5b), whereas a low but definite percentage of these cells were detected in the patients with cGVHD (Figure 5c). The percentages of IL-4-producing CD8+ T cells among CD8+ T cells in each patient were summarized in Table 2. The percentage of IL-4-producing CD8+ T cells in patients with cGVHD was not significantly affected by the use of immunosuppressive agents or corticosteroids, although a patient with cGVHD receiving the agents showed a relatively lower value.

Figure 4
figure4

The percentage of IL-4 and IFN-γ -producing CD8+ T cells by intracellular cytokine staining. The open column is controls (n=10), the dotted column is patients without cGVHD (n=9), and the black column is patients with cGVHD (n=10). **P<0.01. ***P<0.001. (a) The percentage of IL-4-producing CD8+ T cells among total CD8+ T cells. (b) The percentage of IFN-γ-producing CD8+ T cells among total CD8+ T cells.

Figure 5
figure5

Representative IL-4 and IFN-γ double staining of CD8+ T cells. (a) normal control, (b) a patient without cGVHD, (c) a patient with cGVHD.

Table 2 Percentage of IL-4-producing CD8+ T cells (%) in patients with cGVHD by intracellular cytokine staining

Increased IL-4 production from CD8+ T cells in patients with cGVHD

The titer of IL-4 in the supernatants of 24-h-cultured CD8+ T cells was significantly higher in patients with cGVHD (mean 593 pg/ml; range 48–1446 pg/ml) than in patients without cGVHD (mean 109 pg/ml; range 29–1303 pg/ml) (P=0.02) and normal control subjects (mean 168 pg/ml; range 0–497 pg/ml) (P=0.006) (Figure 6a), which was consistent with the results obtained by intracellular cytokine staining. The titer of IL-4 from CD8+ T cells was no different between patients without cGVHD and normal control subjects. On the other hand, the titer of IFN-γ from CD8+ T cells was higher in patients with cGVHD (mean 18209 pg/ml; range 6416–45091 pg/ml) and without cGVHD (mean 18609 pg/ml; range 7210–41292 pg/ml) than in normal control subjects (mean 7898 pg/ml; range 2169–17811 pg/ml) (P=0.004, 0.017) (Figure 6b). However, the titer was not different between the two patient groups.

Figure 6
figure6

The cytokine titer in the supernatants of 24-h-cultured CD8+ T cells. The open column is controls (n=10), the dotted column is patients without cGVHD (n=9), and the black column is patients with cGVHD (n=10). *P<0.05. **P<0.01. (a) The titer of IL-4 from CD8+ T cells. (b) The titer of IFN-γ from CD8+ T cells.

Discussion

In this study, the analysis of cytokine production of peripheral blood CD4+ T cells and CD8+ T cells demonstrated that predominant type 1 cytokine producing helper T cells (Th1) and type 1 cytokine producing cytotoxic T cells (Tc1) balance exist in the patients in the chronic phase of allo-HSCT, irrespective of the presence or absence of cGVHD. These Th1 cells and Tc1 cells might cooperatively exert some cytotoxic effects in the patients, because the Tc1 cells population is known to contain effector cytotoxic CD8+ T cells and Th1 cells support the induction of Tc1 cells.29 Although the target cells of the Th1 cells and Tc1 cells are unknown, it might well be that these cell types are related to the graft-versus-leukemia effect presumed to be operating in the patients in view of the finding that no patients had experienced leukemia relapse or infection at the time of examination.

There is still controversy regarding the Th balance in cGVHD. Some murine studies suggest that Th2 cytokines are associated with cGVHD.19, 22 On the other hand, other studies favor a Th1-driven mechanism of cGVHD in both murine and human systems.23, 24, 25 However, the current study could not detect any significant difference in IL-4 or IFN-γ production from CD4+ T cells between patients with and without cGVHD. Although the reason for the discrepancy in the results between the previous reports and this study is currently unknown, it might be related to the difference in the severity of cGVHD examined. Indeed, although the cGVHD in four patients was initially of the extensive type, basically all cGVHD in this study fell into the limited-type category at the time of examination. In this context, it is critical to examine whether some Th skewing occurs in extensive cGVHD.

One of the most remarkable findings of this study is that the appearance of IL-4-producing CD8+ T cells strictly correlates with the occurrence of cGVHD. In addition, the percentage of IL-4-producing CD8+ T cells in patients with cGVHD was not significantly affected by the use of immunosuppressive agents or corticosteroids. To our knowledge, this is the first report that describes the increase of IL-4-producing CD8+ T cells in cGVHD after allo-HSCT. Although it is established that CD8+ T cells exert cytotoxic effects, the function of IL-4-producing CD8+ T cells in vivo is still unclear. In vitro, IL-4-producing CD8+ T cells are induced from naive CD8+ T cells by administration of IL-4.30, 31 These induced IL-4-producing CD8+ T cells have no cytotoxicity and instead produce type 2 cytokines such as IL-5 and IL-10 in addition to IL-4.31 Furthermore, these induced IL-4-producing CD8+ T cells are shown to have the capacity to induce the production of immunoglobulins from B cells.31, 32 Another report using a murine model suggests that increased serum immunoglobulins, elevated IL-4 synthesis and autoantibody production are closely associated with the pathogenesis of cGVHD.22, 32, 33, 34, 35 Based on these findings, the IL-4-producing CD8+ T cells found in the patients with cGVHD in the current study might have some functional role in inducing cGVHD through type 2 cytokine production, although we could not detect an increase of IL-4-producing CD4+ T cells on the occurrence of cGVHD. Another possibility is that the IL-4-producing CD8+ T cells may be induced as a feedback mechanism to counteract Th1/Tc1 balance in cGVHD, because IL-4 is known to suppress type 1 cytokines such as IFN-γ.31 Thus, it is a critical future work to explore the precise functional role of the IL-4-producing CD8+ T cells in cGVHD. Despite this situation, the IL-4-producing CD8+ T cells may serve as a hallmark that discriminates between the presence and absence of cGVHD.

In this study, the use of immunosuppressive agents or corticosteroids for the purpose of controlling cGVHD did not significantly affect the total number of CD4+ T cells or CD8+ T cells in both patients with and without cGVHD. Furthermore, they did not significantly altered IL-4 and IFN-γ production from CD4+ T cells and CD8+ T cells in both patients with and without cGVHD (data not shown). Thus, it is interesting to know whether the further or extended application of the agents to the patients with cGVHD normalizes the percentage of IL-4-producing CD8+ T cells and ameliorate the symptoms of GVHD. In this context, it is of note that a patient with cGVHD receiving the agents showed a relatively lower percentage of IL-4-producing CD8+ T cells.

In conclusion, we demonstrated in this study that IFN-γ-producing CD4+ T cells and CD8+ T cells were significantly increased in the chronic phase of allo-HSCT, implicating a relevance of these cells with graft-versus-leukemia effect. On the other hand, we also suggest that IL-4-producing CD8+ T cells may be an immunological marker of cGVHD. These findings will provide a better understanding of the immune responses evoked during the chronic phase of allo-HSCT.

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Acknowledgements

We thank Professor Seizaburo Arita of Kansai Medical University for the evaluation of statistical analyses. We also thank Miss Tomoko Masuda for her secretarial work and assistance in experiments.

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Correspondence to R Amakawa.

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Nakamura, K., Amakawa, R., Takebayashi, M. et al. IL-4-producing CD8+ T cells may be an immunological hallmark of chronic GVHD. Bone Marrow Transplant 36, 639–647 (2005) doi:10.1038/sj.bmt.1705107

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Keywords

  • IL-4-producing CD8+ T cells
  • chronic GVHD
  • HSCT
  • cytokines

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