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Keywords:

  • calcineurin inhihitor;
  • early post-transplantaion period;
  • human;
  • liver transplantation;
  • regulatory T cell;
  • T helper type 17 cells

Summary

  1. Top of page
  2. Summary
  3. Introduction
  4. Patients and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Authorship
  9. Disclosure
  10. References

There is limited clinical research regarding the changes in peripheral lymphocyte subsets during the early post-operative period of liver transplantation. Serial changes of T cells and B cells in living donor liver transplantation (LDLT) recipients during the early post-transplantion period were prospectively investigated. From June 2010 to February 2011, 27 consecutive LDLT recipients were enrolled. Percentages of T helper type 1 (Th1; interferon-γ-producing), Th2 (interleukin-4-producing), Th17 (interleukin-17-producing), regulatory T (Treg; CD4+ CD25+ FoxP3+), memory B (CD19+ CD24hi CD38) and mature B (CD19+ CD24int CD38int) cells were measured using fluorescence-activated cell sorting. Patients were followed up for a median of 9·9 months (range 6·8−15·5 months) after transplantation. Serial monitoring of immunological profiles showed no significant suppression of Th1, Th2, Th17, mature B or memory B cells, whereas frequencies of Treg cells significantly decreased. Interleukin-17 production by central and effector memory cells was not suppressed during the early post-operative period. The continuous production of interleukin-17 by the memory T cells may contribute to the persistence of Th17 cells. This prospective study demonstrated that current immunosuppression maintained the effector T or memory B cells during the early post-transplantation period but significantly suppressed Treg cells. Serial immune monitoring may suggest clues for optimal or individualized immunosuppression during the early post-operative period in clinical practice.


Abbreviations
APC

allophycocyanin

Cy7

Cychrome 7

IL

interleukin

LDLT

living donor liver transplantation

PBMC

peripheral blood mononuclear cells

PE

phycoerythrin

TCM

central memory T cell

TEM

effector memory T cell

Th17 cells

T helper 17 cells

Treg cells

regulatory T cells

Introduction

  1. Top of page
  2. Summary
  3. Introduction
  4. Patients and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Authorship
  9. Disclosure
  10. References

In recent years, the incidence of early allograft loss caused by acute rejection has decreased with the development of potent immunosuppressive agents such as cyclosporin A, tacrolimus and mycophenolic acid. Induction with a combination of potent immunosuppressants became widely accepted as a method of minimizing the risk of acute rejection, which may be clinically fatal in the process of liver transplantation (LT).[1-3] However, intensive immunosuppression has been documented to increase the risk of infections, malignancies and post-transplant lymphoproliferative disorders.[4, 5] Moreover, the immunosuppressive therapies that are currently used during the early post-transplantation period target all of the immune responses, which may have deleterious effects on the regulatory mechanisms responsible for tolerance induction.[6] Immunological monitoring during the early post-operative period after LT may be beneficial to detect rejection faster by applying individually tailored immunosuppression protocols.[7] Several investigators have studied serial changes in circulating cytokine levels during the early post-operative period from the perspective of the T helper type 1 (Th1)/Th2 paradigm. However, the results of these studies have been inconsistent.[7-11]

Recently identified regulatory T (Treg) cells and interleukin-17 (IL-17) -producing CD4+ T cells (Th17) are key suppressors and effectors in the immune response, respectively. The Treg cells have been reported to be involved in transplant tolerance, whereas Th17 cells have essential roles in transplant rejection.[12] It has been suggested that Th17 cells perform a role reciprocal to that of Treg cells,[12-14] and serve as B-cell helpers in that they not only induce B-cell proliferation in vitro, but also promote antibody production with class-switch recombination in vivo.[15] However, there have been no data illustrating the dynamics and interactions between Th17, Treg and B cells during the early post-operative period in a clinical LT setting.

The host immune response to graft liver is vigorous within several weeks,[16] so the intensity of immunosuppression in most transplantation centres is potent during the early post-operative period (within 3–4 weeks) but then gradually decreases.[17] The exact immune status of patients during the early post-transplantation period is an interesting issue from the point of view of the balance between over-immunosuppression and under-immunosuppression.

We hypothesized that the current immunosuppression would target all of the immune responses non-specifically. Therefore, both regulatory and effector cells would be suppressed in LT recipients with intensive immunosuppression during the early post-transplantation period. The aim of this study was to investigate the changes of T cells and B cells in the peripheral blood of living donor liver transplantation (LDLT) recipients during the early post-transplantation period under the influence of intensive immunosuppression.

Patients and methods

  1. Top of page
  2. Summary
  3. Introduction
  4. Patients and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Authorship
  9. Disclosure
  10. References

Patient recruitment and clinical samples

Between June 2010 and February 2011, 47 adult LDLT were performed at the liver transplantation centre. Patients with a recent infection before transplantation (n = 1), ABO-incompatible LT (n = 3), lack of consent to participate in the study (n = 5), and lack of appropriate blood samples (n = 11) were excluded. The remaining 27 consecutive LDLT patients were recruited prospectively and underwent immune monitoring before undergoing the LT process and during the first 3 weeks after transplantation.

All patients received a standard triple immunosuppressive therapy consisting of corticosteroids, calcineurin inhibitors [either tacrolimus (n = 23) or cyclosporin A (n = 4)] and mycophenolate mofetil. Methylprednisolone (10 mg/kg) was administered intravenously immediately before reperfusion and continued for 7 days. This was then switched to an oral administration of prednisolone at a dose of 0·3 mg/kg. The dosage of calcineurin inhibitors was adjusted to target the serum trough level of tacrolimus of 5–10 ng/ml or to maintain the serum level of cyclosporin A at 200–250 ng/ml. Mycophenolate mofetil (500 mg) was administered orally twice daily.

Blood samples were serially collected on the day before LDLT (pre-transplant) and on days 7, 14 and 21 after transplantation. Thirty-two age-matched healthy blood donors were tested as controls. The patients and healthy controls provided their written informed consent. The study was conducted according to the current declaration of Helsinki and the protocol was approved by the institutional ethics committee of Seoul St Mary's Hospital (KC10TISI0433).

FACS analysis

Peripheral blood mononuclear cells (PBMC) were isolated from heparinized venous blood by standard density gradient centrifugation over Ficoll-Paque (GE Healthcare Biosciences, Uppsala, Sweden). The PBMC were stimulated with 50 ng/ml PMA (Sigma-Aldrich, St Louis, MO) and 1 μg/ml ionomycin (Sigma-Aldrich) and Golgi Stop (BD Biosciences, San Diego, CA) were added for 4 hr. The cells were washed and 5 × 105 cells per sample were incubated for surface markers for 30 min at 4° in the dark. The cells were then permeabilized using a Cytofix/Cytoperm Plus kit (BD Biosciences) and stained with antibodies specific for intracellular markers for 30 min at 4° in the dark. For analysis of Treg cells, PBMC were surface labelled with CD4 and CD25, followed by fixation, permeabilization and intracellular staining with FoxP3. Treg-cell staining was performed using the eBioscience FoxP3 staining kit (eBioscience, San Diego, CA).

Antibodies used for surface analysis

The following monoclonal antibodies were used: phycoerythrin (PE)/Cyanine 7 (Cy7)-conjugated anti-CD4 (Biolegend, San Diego, CA), FITC-conjugated anti-CD45RA (Pharmingen, San Diego, CA), allophycocyanin (APC)-conjugated anti-CD25 (Pharmingen), peridinin chlorophyll protein (PerCP)-Cyanine 5.5(Cy5.5)-conjugated anti-CD38 (Pharmingen), FITC-conjugated anti-CD19 (Southern Biotech, Birmingham, AL), PE-conjugated anti-CD24 (Pharmingen), and APC-conjugated anti-Annexin V (Invitrogen, Grand Island, NY).

Antibodies used for chemokine receptors

The following mouse monoclonal antibody was used: anti-CCR7 (Pharmingen).

Antibodies used for intracellular cytokines

Phycoerythrin-conjugated anti-IL-17 (eBioscience), FITC-conjugated anti-interferon-γ (eBioscience), APC-conjugated anti-IL-4 (eBioscience), FITC-conjugated anti-FoxP3 (eBioscience). Appropriate isotype controls were used for gate setting for cytokine expression. Cells were analysed using a FACSCalibur flow cytometry system (Becton Dickinson Systems, BD Biosciences, San Jose, CA) and FlowJo software (Tree Star, Ashland, OR).

Cell culture

Cell cultures were performed in a RPMI-1640 medium (GibcoBRL, Carlsbad, CA) containing penicillin (100 U/ml), streptomycin (100 μg/ml) and 10% fetal bovine serum (GibcoBRL) that became inactivated when heated to 55° for 30 min. The cell suspensions were dispensed into 48-well plates (Nunc, Roskilde, Denmark). Cells were activated at a concentration of 5 × 106/500 μl medium with anti-CD3 (1 μg/ml) and anti-CD28 (1 μg/ml) for 72 hr. Th-neutral conditions (Th0) contained no exogenous cytokines or anti-cytokines.

Immunofluorescence analysis

A 100-μl aliquot of each sample was put into the appropriate well of a cytospin chamber (Thermo Scientific, Michigan, MI) and was centrifuged at 800 g for 3 min at 4°. To preserve the membrane-associated components and foreclose cytoplasmic staining, cells were fixed with methanol–acetone at −20° for 10 min. Immunofluorescence investigations were carried out using the PE-conjugated anti-RORγt (1 : 100; BD Biosciences), FITC-conjugated anti-IL-17 (1 : 100; BD Biosciences) and PerCP-Cy5.5-conjugated anti-CD4 (1 : 50; BD Biosciences) and 4′,6-diamidino-2-phenylindole (DAPI). The stained sections were analysed using a Zeiss microscope (LSM 510 Meta; Carl Zeiss, Oberkochen, Germany) at 400 × magnification.

Measurement of cytokines

Cytokine concentrations of IL-6 and IL-22 in cell culture supernatants were analysed by ELISA. Antibodies directed against human IL-6 and IL-22 and against biotinylated anti-human IL-6 and IL-22 were used as the capture and detection antibodies, respectively. Alkaline phosphatase (Sigma) was used for the chromogenic reaction. The amounts of cytokines present in the test samples were determined from standard curves constructed with serial dilutions of recombinant human IL-6 and IL-22 (R&D Systems, Minneapolis, MN). The absorbance was determined with an ELISA microplate reader at 405 nm.

In vitro suppression assays

Peripheral blood mononuclear cells were cultured with anti-CD3 (1 μg/ml), anti-CD28 (1 μg/ml) and calcineurin inhibitor for 24 hr. Total RNA was prepared from the cultured cells and was extracted using TRIzol (Molecular Research Center, Cincinnati, OH). Two micrograms of total RNA was reverse transcribed using the Superscript Reverse Transcription system (Takara, Shiga, Japan). The levels of mRNA expression were estimated using real-time quantitative PCR with LightCycler FastStart DNAmaster SYBR green I (Takara), according to the manufacturer's instructions.

The primer pairs used in these reactions were as follows:

  • control for human β-actin:
  • forward 5′-GGACTTCGAGCAAGAGATGG-3′,
  • reverse 5′-TGTGTTGGCGTACAGGTCTTTG-3′;
  • for IL-2:
  • forward 5′-GAATGGAATTAATAATTACAAGAATCC-3′,
  • reverse 5′-TGTTTCAGATCCCTTTAGTTCCAG-3′.

The amplification reactions, data acquisition and analysis were performed with the LightCycler Real-Time PCR system (Roche Diagnostics, Mannheim, Germany), and relative levels of gene expression were normalized against β-actin.

To examine the expression of phospho-STAT5 following IL-2 exposure, human PBMC were pre-treated with 20 ng/ml recombinant IL-2 for 1 hr. Cells were stained with PerCP-conjugated anti-CD4 antibody (Pharmingen), PE-conjugated anti-CD25 (Pharmingen), followed by FITC-conjugated anti-phospho-STAT5 (Y694) (all from Pharmingen) using the Fixation Buffer and Perm Buffer III (Pharmingen) according to the manufacturer's instructions.

Statistical analysis

Continuous data were expressed as the mean ± standard deviation and were compared using the Mann–Whitney U-test. Categorical data were expressed as the number of patients. Friedman's test was used for analysis of repeated measures. Post hoc analysis using the Wilcoxon signed-rank test was conducted and a Bonferroni correction was applied. A P-value less than 0·05 was considered significant. SPSS version 14 software (SPSS, Chicago, IL) was used.

Results

  1. Top of page
  2. Summary
  3. Introduction
  4. Patients and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Authorship
  9. Disclosure
  10. References

Baseline clinical characteristics of the enrolled patients are listed in Table 1. Patients were followed up for a median of 9·9 months (range 6·8–15·5 months) after transplantation. None of these patients developed acute cellular rejection within 1 month after LDLT. All recipients maintained good graft function in the early post-transplantation period. After the early post-operative period, biopsy-proven rejection with a Banff rejection activity index of 7 occurred in one patient (5 months after transplantation).

Table 1. Baseline characteristics of the study population
Variablen = 27
  1. Continuous data are expressed as the mean values ± standard deviation.

  2. LC, liver cirrhosis; HCC, hepatocellular carcinoma; HBV, hepatitis B virus; MELD, model for end-stage liver disease.

Sex, n
Female/Male8/19
Age, years51·7 ± 8·2
Primary disease, n
LC/HCC17/10
Aetiology, n
HBV/Alcohol/others17/5/5
Child–Pugh class, n
A/B/C9/7/11
MELD score11·3 ± 6·7

Comparison of Th1, Th2, Th17, Treg and B cells between pre-transplant patients and controls

Figure 1(a) shows the frequency of interferon-γ-producing Th1 cells, IL-17-producing Th17 cells, IL-4-producing Th2 cells, CD4+ CD25+ FoxP3+ Treg cells, CD19+ CD24hi CD38 memory B cells, and CD19+ CD24int CD38int mature B cells in both patients and controls.

image

Figure 1. (a) Representative examples of flow cytometry results for a patient before transplantation and for a control. Results for interferon-γ-producing and interleukin-17 (IL-17) -producing T lymphocytes [T helper type 1 (Th1) and Th17], IL-4-producing T lymphocytes (Th2), CD4+ CD25+ FoxP3+ T lymphocytes (Treg), CD19+ CD24hi CD38 (memory B), CD19+ CD24int CD38int (mature B) B cells are shown. (b) Immunofluorescence analysis of peripheral blood mononuclear cells (PBMC) in the patients and the controls. CD4+ cells (white) co-expressing IL-17 (green) and RORγt (red) were not significantly different between the patients and controls. (c) Production of Th17 signature cytokines. The amounts of IL-6 and IL-22 under Th0 culture conditions were significantly higher in cell culture supernatants of the patients compared with those of the controls.

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There were no statistically significant differences of baseline frequencies of the subsets of Th1, Th2 and Treg cells between the patients and controls (Table 2). However, the proportion of Th17 cells was significantly higher in the patient group compared with the control group (patients, 0·89 ± 0·32%; control, 0·71 ± 0·25%; = 0·022). As shown in Fig. 1(b), the frequency of cells expressing RORγt was significant higher in the patients than in the controls. CD4 T cells co-expressing IL-17 and RORγt differ between the patients and controls. Both IL-6 and IL-22, Th17 signature cytokines that were under Th0 culture conditions, were significantly higher in the cell culture supernatants of the patients compared with those of the controls (Fig. 1c). The frequency of memory B cells was lower in the patient group than in the control group (patients, 11·7 ± 10·9%; control, 25·4 ± 6·4%; < 0·001), whereas the percentage of mature B cells was higher in the patient group than in the control group (patients, 69·7 ± 11·4%; control, 57·3 ± 6·7%; < 0·001).

Table 2. Percentages of lymphocyte subsets in the patient group compared with the control group
 Th11Th21Th171Treg1Memory B2Mature B2
  1. a

    Mann–Whitney U-test. Data are expressed as the mean ± standard deviation.

  2. 1% within CD4+ T cells.

  3. 2% within CD19+ B cells.

  4. Th1: interferon-γ-producing CD4+ T cell; Th2: interleukin-4-producing CD4+ T cell; Th17: interleukin-17-producing CD4+ T cell; Treg: CD4+ CD25+ + regulatory T cell; memory B cell: CD19+ CD24hi CD38 B cell; mature B cell: CD19+ CD24int CD38int B cell.

Control (n = 32)10·1 ± 5·24·3 ± 1·80·71 ± 0·253·8 ± 1·125·4 ± 6·457·3 ± 6·7
Patient (n = 27)11·7 ± 7·23·8 ± 1·50·89 ± 0·323·8 ± 1·311·7 ± 10·969·7 ± 11·4
P valuea0·3940·1550·0220·493< 0·001< 0·001

Effector T cells were maintained and regulatory T cells were decreased during the early post-transplantation period

The percentage of CD4 T cells decreased 1 week after transplantation (pre-transplant, 42·8 ± 13·5%; Day 7, 36·1 ±13·4%; Day 14, 34·2 ± 13·4%; Day 21, 37·1 ± 10·9%; = 0·001) (Fig. 2a). The trough level of calcineurin inhibitors rapidly increased after LT and reached the target range between 14 and 21 days after transplantation (Fig. 2b).

image

Figure 2. Serial changes of (a) percentage of CD4+ T cells and (b) trough levels of calcineurin inhibitors in recipients during the early post-operative period after liver transplantation. Values are expressed as means. The bars show standard deviations. D7: 7 days after transplantation; D14: 14 days after transplantation; D21: 21 days after transplantation.

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With the exception of one case of rejection in the early post-transplantation period, serial monitoring of the immunological profiles demonstrated no significant changes in the percentages of effector T cells, such as Th1 (= 0·053) (Fig. 3a), Th2 (= 0·671) (Fig. 3b), or Th17 cells (pre-transplant, 0·90 ± 0·32%; Day 7, 0·91 ± 0·39%; Day 14, 0·74 ± 0·32%; Day 21, 0·75 ± 0·38%; = 0·443) (Fig. 3c), regardless of treatment with immunosuppressive agents. However, when immunosuppressants were introduced, the percentage of Treg cells significantly decreased (pre-transplant, 3·59 ± 1·17%; Day 7, 1·72 ± 0·68%; Day 14, 1·63 ± 1·03%; Day 21, 1·33 ± 0·48%; < 0·001) (Fig. 3d). In terms of B cells, the percentage of memory B cells significantly increased at 1 week after the transplant (pre-transplant, 8·76 ± 5·12%; Day 7, 12·76 ± 11·63%; Day 14, 12·56 ± 8·7%; Day 21, 12·59 ± 7·69%; = 0·002) (Fig. 3e), whereas the frequency of mature B cells showed little change (Fig. 3f). Results for Th17, Treg and memory B cells in the peripheral blood before transplantation and on day 7 using flow cytometry are shown in Fig. 4.

image

Figure 3. Serial changes in T lymphocyte and effector B-cell subsets during early post-operative period after liver transplantation. There were no significant differences in the proportions of T helper type 1 (Th1) (a), Th2 (b) and Th17 cells (c); however, the frequency of regulatory T (Treg) cells (d) decreased. The proportion of memory B cells (e) increased, whereas the frequency of mature B cells (f) showed little change in the early post-transplantation period. Values are expressed as means. The bars show standard deviations. D7: 7 days after transplantation; D14:  14 days after transplantation; D21: 21 days after transplantation.

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image

Figure 4. Flow cytometry analysis of T helper type 17 (Th17), regulatory T (Treg) and memory B cells in the peripheral blood before transplantation and on day 7 thereafter. Representative flow cytomety results are shown, and the percentage of each subset is indicated in the dot plots. D7: 7 days after transplantation.

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Blocking IL-2 signalling pathway may contribute to the decrease in Treg cells in patients receiving calcineurin inhibitors

Expression of annexin V was not significantly different between the patients and the healthy controls (Fig. 5a). Expression of IL-2 mRNA was markedly reduced in patients receiving calcineurin inhibitors (Fig. 5b). Furthermore, the expression of phospho-STAT5 following IL-2 exposure was significantly less elevated in patients taking calcineurin inhibitors compared with controls (Fig. 5c).

image

Figure 5. Blocking of interleukin-2 (IL-2) signalling pathway by calcineurin inhibitors may contribute to the decrease of regulatory T (Treg) cells. (a) Annexin V staining in Treg cells in the peripheral blood of patients and controls. (b) Inhibition of IL-2 expression by calcineurin inhibitors. CD4+ CD25+ cells isolated by flow cytometry were stimulated for 24 hr with anti-CD3 and anti-CD28 under neutral conditions with FK506. IL-2 expression normalized to β-actin levels was markedly reduced in patients after treatment with calcineurin inhibitors. (c) Phospho-STAT5 expression following IL-2 exposure. IL-2-induced STAT5 phosphorylation was significantly decreased in patients receiving calcineurin inhibitors.

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Central memory T cells increased as effector memory T cells were maintained during the early post-transplantation period

Staining of peripheral blood T cells with antibodies to CD45RA and CCR7 divided two memory subpopulations of CD4+ T cells: CD45RA CCR7+ central memory T cells (TCM) and CD45RA CCR7 effector memory T cells (TEM).

The TCM percentage increased 1 week after transplantation (pre-transplant, 50·9 ± 11·7%; Day 7, 56·9 ± 11·0%; Day 14, 52·7 ± 11·2%; Day 21, 54·8 ± 11·4%; = 0.003) (Fig. 6a). The percentage of TEM did not show any significant changes over time during the early post-transplant period (= 0·468) (Fig. 6b). Within the two CD4+ memory T-cell subsets (TCM and TEM), IL-17 production showed little change (Fig. 6c,d).

image

Figure 6. Serial changes in central memory (TCM) and effector memory (TEM) T-cell subpopulations of CD4+ T lymphocytes during the early post-operative period after liver transplantation. (a) CD45RA CCR7+/CD4+ T cells (TCM/CD4+ T) and (b) CD45RA CCR7/CD4+ T cells (TEM/CD4+ T cells). Serial changes in interleukin-17 (IL-17) in CD4+ T-cell subsets by intracellular flow cytometry. (c) CD45RA CCR7+ IL-17+/CD4+ T cells (TCM IL-17+/CD4+ T) and (d) CD45RA CCR7 IL-17+/CD4+ T cells (TEM IL-17+/CD4+ T cells). Values are expressed as means. The bars show standard errors of the means. *< 0·017. D7: 7 days after transplantation; D14: 14 days after transplantation; D21: 21 days after transplantation.

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Discussion

  1. Top of page
  2. Summary
  3. Introduction
  4. Patients and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Authorship
  9. Disclosure
  10. References

The intensity of immunosuppression is important in the early post-operative period because the host immune response to graft liver becomes vigorous within 3–4 weeks post-transplantation.[16] Therefore, the investigation to determine the exact immune status of patients during the early post-transplantation period is of interest. This prospective study of immunological profiles interestingly demonstrated that Treg cells, a well-known tolerance marker, significantly decreased with little suppression of the subsets of Th1, Th2, Th17 and effector B cells upon introducing conventional immunosuppression immediately after LDLT.

Specifically, calcineurin inhibitors selectively block the expression of the IL-2 gene, which is the signal required for the survival and function of Treg cells. Several studies demonstrated that the numbers of Treg cells were decreased with the use of calcineurin inhibitors.[18, 19] Similarly our results showed that the proportion of anti-inflammatory Treg cells substantially decreased. However, tissue-destructive effector Th17 cells were not suppressed despite the potent immunosuppression in the early post-operative period. As demonstrated in Fig. 5(b,c), suppression of IL-2 expression by calcineurin inhibitors and subsequent blocking of the IL-2 pathway on Treg cells may contribute to the suppression of Treg cells.

Th17 cells have been considered to play a role in allograft rejection in the context of organ transplantation.[20-22] However, there is limited knowledge regarding the dynamics of Th17 cells in LDLT recipients under immunosuppression during the early post-transplantation period. Current immunosuppressants target both pro-inflammatory and protective T cells non-specifically[23] so Th17 cells as well as Treg cells have been reported to be down-regulated upon immunosuppression. However, there are conflicting results on whether Th17 cells are suppressed by calcineurin inhibitors or mycophenolic acid.[24-27] In vitro, cyclosporin A inhibited IL-17 transcription and mRNA expression in PBMC from patients with rheumatoid arthritis.[27-29] In contrast, other studies reported that IL-17 production was not inhibited by cyclosporin A or tacrolimus in activated human PBMC.[24-26] In terms of mycophenolic acid, one study showed that IL-17 transcription was inhibited by mycophenolic acid in activated human PBMC,[24] but others have reported findings that have contrasted this.[26] Our in vivo study demonstrated the maintenance of Th17 cells in the peripheral blood of patients during the early post-transplantation period after LDLT. Notably, the frequency of Th17 cells would increase upon liver transplantation without the immunosuppressants, hence showing enough of an effect to suppress the increase in Th17 cells.

Memory T cells may interfere with the induction of tolerance to allografts.[30] In our study, the initial increase seen in TCM cell percentages may be associated with the allograft stimuli immediately after transplantation. Additionally, the IL-17 production by TCM or TEM cells was not suppressed during the early post-operative period. The continuous production of IL-17 by memory T cells may contribute to the persistence of the Th17 cells. This study also showed that the percentage of memory B cells with effector function increased during the early post-operative period. As Th17 cells can induce effector B-cell differentiation,[15] uncontrolled Th17 cells regardless of any immunosuppression, might contribute to the proliferation of effector B cells.

We experienced only one case of acute rejection at 5 months after LDLT. The trough level of calcineurin inhibitor was maintained at a similar serum level compared with other patients. It is important to note that this patient displayed a decrease in the frequency of Treg cells upon starting immunosuppression in a manner similar to that of the other recipients during the early post-operative period, whereas the proportion of Th17 cells increased 3 weeks post-transplant. Suppression of anti-inflammatory Treg cells by immunosuppressants might have resulted in an increase in the numbers of pro-inflammatory Th17 cells without a counterbalance. Although one case was available because of the lack of rejection episodes, it suggested a possibility that the optimal level of immunosuppression would be attained when Th17 cells were suppressed, but the proportion of regulatory cells was maintained.

To our knowledge, this is the first prospective study on the dynamic changes of Treg, Th17 and B-cell responses under current immunosuppressive agents during the early post-transplantation period. However, there are several limitations. First, intra-operative and post-operative factors could have affected the immunological response. During the study period, no serious incidents of infection, bleeding or biliary complications were noted. Therefore, surgical or inflammatory factors would have influence the immunological profiles less. Second, the immunological profiles were not investigated in the liver tissues because performing a liver biopsy would have increased the risk of bleeding during the early post-transplantation period. Generally, the immune status in the peripheral blood would be different from that of the liver tissues. Lastly, we investigated the short-term immunological responses during the 3 weeks after LDLT. However, the early post-operative period is the critical time when the host immune system responds to a graft liver under immunosuppression as reflected by the observation that the surge of migratory donor mononuclear leucocytes appears in the recipient blood during the first 2 or 3 weeks after the intestinal transplantation.[16]

In conclusion, the results of this prospective study indicate that under conventional immunosuppression protocols, the percentage of T cells with regulatory properties decreased in the early post-transplantation period, and the percentages of effector Th17 cells were unaffected. In current clinical practice, the serum level of calcineurin inhibitors is the only surrogate marker used to modify the intensity of immunosuppression after LDLT. These results suggest that immune monitoring during the early post-operative period may provide initial signs for optimal or individualized immunosuppression.

Acknowledgements

  1. Top of page
  2. Summary
  3. Introduction
  4. Patients and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Authorship
  9. Disclosure
  10. References

This research was supported by a grant from the Korea Health Technology R&D Project, Ministry for Health, Welfare & Family Affairs, Republic of Korea (grant number A092258) and was partly supported by a grant from the Korean Healthcare Technology R&D Project, Ministry for Health and Welfare, Republic of Korea (grant number A102065).

Authorship

  1. Top of page
  2. Summary
  3. Introduction
  4. Patients and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Authorship
  9. Disclosure
  10. References

HYK and MLC: analysed data and wrote the manuscript. JYJ and JKB: performed the research. BHC and CWY: contributed important concepts. SKY, SHB and DGK: collected the data. JYC: designed the research and wrote the manuscript.

References

  1. Top of page
  2. Summary
  3. Introduction
  4. Patients and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Authorship
  9. Disclosure
  10. References