Analyses of Peripheral Blood Mononuclear Cells in Operational Tolerance After Pediatric Living Donor Liver Transplantation

Authors

  • Ying Li,

    1. Department of Transplantation and Immunology and Horizontal Medical Research Organization, Transplant Tolerance Unit, Kyoto University, Faculty of medicine, Kyoto, Japan
    Search for more papers by this author
  • Takaaki Koshiba,

    1. Department of Transplantation and Immunology and Horizontal Medical Research Organization, Transplant Tolerance Unit, Kyoto University, Faculty of medicine, Kyoto, Japan
    Search for more papers by this author
  • Atsushi Yoshizawa,

    1. Department of Transplantation and Immunology and Horizontal Medical Research Organization, Transplant Tolerance Unit, Kyoto University, Faculty of medicine, Kyoto, Japan
    Search for more papers by this author
  • Yukihide Yonekawa,

    1. Department of Transplantation and Immunology and Horizontal Medical Research Organization, Transplant Tolerance Unit, Kyoto University, Faculty of medicine, Kyoto, Japan
    Search for more papers by this author
  • Kosuke Masuda,

    1. Department of Transplantation and Immunology and Horizontal Medical Research Organization, Transplant Tolerance Unit, Kyoto University, Faculty of medicine, Kyoto, Japan
    Search for more papers by this author
  • Atsushi Ito,

    1. Department of Transplantation and Immunology and Horizontal Medical Research Organization, Transplant Tolerance Unit, Kyoto University, Faculty of medicine, Kyoto, Japan
    Search for more papers by this author
  • Mikiko Ueda,

    1. Department of Transplantation and Immunology and Horizontal Medical Research Organization, Transplant Tolerance Unit, Kyoto University, Faculty of medicine, Kyoto, Japan
    Search for more papers by this author
  • Takahide Mori,

    1. Daigo Watanabe Clinic, 30–15 Takahata-cho Daigo Fushimiku, 601–1375, Kyoto, Japan
    Search for more papers by this author
  • Hiroshi Kawamoto,

    1. Laboratory of Immunology and Cell Biology and Laboratory for Lymphocyte Development, RIKEN RCAI, Graduate School of Biostudies, Kyoto University
    Search for more papers by this author
  • Yoshimasa Tanaka,

    Corresponding author
    1. Laboratory of Immunology and Cell Biology and Laboratory for Lymphocyte Development, RIKEN RCAI, Graduate School of Biostudies, Kyoto University
    Search for more papers by this author
  • Shimon Sakaguchi,

    1. Department of Experimental pathology, Institute for Frontier Medical Sciences, Kyoto University, Kyoto, Japan
    Search for more papers by this author
  • Nagahiro Minato,

    1. Laboratory of Immunology and Cell Biology and Laboratory for Lymphocyte Development, RIKEN RCAI, Graduate School of Biostudies, Kyoto University
    Search for more papers by this author
  • Kathryn J. Wood,

    1. Nuffield Department of Surgery, University of Oxford, John Radcliffe Hospital, Headington, Oxford, UK
    Search for more papers by this author
  • Koichi Tanaka

    1. Department of Transplantation and Immunology and Horizontal Medical Research Organization, Transplant Tolerance Unit, Kyoto University, Faculty of medicine, Kyoto, Japan
    Search for more papers by this author

*Corresponding authors: Koichi Tanaka, koichi@kuhp.kyoto-u.ac.jp, Takaaki Koshiba, tkoshiba@kuhp.kyoto-u.ac.jp

Abstract

Operational tolerance (graft acceptance in an immunosuppression (IS)-free environment) after living-donor liver transplantation (LDLT) could occur by our elective protocol in some patients. There is, nevertheless, no reliable parameter to monitor patients who may discontinue IS without a risk of rejection. To identify such parameters, we systemically phenotyped peripheral blood mononuclear cells from operationally tolerant patients. An increase was observed in the frequency of CD4+CD25high+ cells, B cells and Vδ1/Vδ2 γδT-cells ratio in operationally tolerant patients (Gr-tol; n = 12), compared with those from age-matched volunteers (Gr-vol; n = 24) or patients on IS (Gr-IS; n = 19). The frequency of NK cells was decreased in Gr-tol, compared with those in Gr-IS or Gr-vol. The frequency of NKT cells was decreased after LDLT, compared with that in Gr-vol. Although the contribution of those subsets to the tolerant state remains elusive, the results may provide important clues for reliable indicators of tolerance after LDLT.

Abbreviations: 
ALT

alanine transaminase

APC

allophycocyanin

AST

aspartate transaminase

BA

biliary atresia

FACS

fluorescence-activated cell sorting

FITC

fluorescein isothiocyanate

IEL

intestinal intraepithelial lymphocyte

IS

immunosuppressant, immunosuppression

LDLT

living-donor liver transplantation

LTx

liver transplantation

NK cells

natural killer cells

NKT cells

natural killer T cells

PBMC

peripheral blood mononuclear cells

PE

phycoerythrin

PTLD

post-transplant lymphoproliferative disorder

Treg

regulatory T cells

Tx

transplant, transplantation

Introduction

Despite the availability of potent immunosuppressive agents including cyclosporine A (CsA), FK506, mycophenolate mofetil (MMF) and rapamycin, acute and chronic rejection remain the leading causes of graft loss after solid organ transplantation (Tx) (1). In addition, long-term exposure to pharmacological immunosuppression (IS) may cause infections, post-transplant lymphoproliferative disorder (PTLD) and malignancies, which contribute significantly to post-Tx morbidity (2). Thus, the achievement of operational tolerance to donor alloantigens, defined as donor-specific graft acceptance in an IS-free environment (3), would lead to a major progress in clinical transplantation.

Experimentally, various mechanisms for the induction and (or) maintenance of tolerance after liver transplantation (LTx) have been proposed, including clonal deletion, anergy of alloreactive T cells and involvement of immunomodulatory leucocytes (4). Recently, there has been extensive interest in the role of regulatory/suppressor cells, whose existence was originally postulated 30 years ago (5). Although the exact nature of regulatory/suppressor cell involved remains elusive, several types of cells have been shown to be tolerogenic in different experimental situations (5). For instance, CD4+CD25+ regulatory T cells (Treg) have been shown to contribute to the establishment of tolerance to alloantigens in rodents (5). In addition, other experimental studies have demonstrated alternative possibilities and suggested that CD8+ cells (6), γδ T cells (7) and NKT cells (8) may also have immuno-regulatory activities that facilitate the development of tolerance. In rodents, spontaneous acceptance (with no need for IS) of liver allografts was often accompanied by substantial graft infiltration by NKT cells (8), which possessed tolerogenic properties (8). Also, Okabe et al. reported that a certain type of γδ T cell, which shared a similar phenotype with intestinal intraepithelial lymphocytes (IEL) and produced Th2-type cytokines such as IL-4 and IL-10, was associated with acceptance of rat hepatic allografts induced by donor-specific blood transfusion (DSBT) (7).

Cases of tolerance after clinical organ Tx are still extremely rare (9). However, in LTx patients, the development of elective protocols has enabled some patients to be weaned off IS (10). We have developed a strategy that enables a substantial proportion (approximately 40%) of LTx patients to be weaned from IS completely. Although this strategy is very successful, some patients (approximately 25%) encountered rejection during the process of weaning from tacrolimus and required the reintroduction of steroids (10). Therefore, investigating the mechanisms responsible for this state of operational tolerance after LTx is very important.

At present, there are no reliable immunological parameters that enable patients who can be weaned from IS without the risk of rejection to be identified. In an attempt to obtain clues that would enable us to define such immunological parameters, which characterize operational tolerance after LDLT, we analyzed systemically lymphocyte subsets present in peripheral blood mononuclear cells (PBMC) from 12 LDLT patients weaned off IS. Our results revealed several intriguing features in the profiles of PBMC in operationally tolerant pediatric LDLT patients that were distinct from those of age-matched control volunteers with normal liver function and patients on IS.

Materials and Methods

Patients

Operational tolerance was defined as acceptance of liver graft for more than 1 year after cessation of IS (4 ± 2 years: 1−6 years). Twelve liver transplantation patients, who fulfilled these criteria, (Group-tol [Gr-tol], 2 males and 10 females, with a mean age of 12 years: 4–23 years) were included in this study. Drug withdrawal was initiated either by the physician-controlled IS weaning protocol (protocol weaning off IS) or for the management of post-Tx infection (cholangitis, Epstein-Barr virus [EBV], hepatitis B virus [HBV]) or PTLD, or as a result of patient non-compliance (non-protocol). Patients with ongoing EBV and HBV infection were not included in this study. Although liver biopsy was not performed because of an unforeseen risk for the recipients, we found that bio-chemical analyses of the peripheral blood showed near normal liver function in all the Gr-tol patients.

Nineteen age-matched LDLT patients on IS group (Group-IS [Gr-IS], 9 males and 10 females, with a mean age of 10 years: 4–24 years) and 24 age-matched outpatient volunteers on vol-group (Group-vol [Gr-vol], 14 males and 10 females, with a mean age of 11 years: 5–23 years) were used as control. In Gr-IS, tacrolimus or CsA-based IS was either maintained because of concern for rejection or was being weaned off. Gr-vol patients were followed at the Department of Pediatrics, Kyoto University Hospital, due to congenital cardiac disease. Except for mild cardiac disease, Gr-vol patients showed no evidences of other diseases and their liver function was normal.

All the LDLT patients and volunteers were examined for complete blood count (CBC), C-reactive protein (CRP) and liver function (aspartate transaminase [AST], alanine transaminase [ALT], total-bilirubin [T-Bil]).

The Ethical Committee of the Faculty of Medicine in Kyoto University approved this study. Informed consent was provided according to the declaration of Helsinki (11). Parental permission was obtained for patients who were younger than 20 years of age.

Isolation of PBMC and cord blood

Venous blood samples (5–10 mL) were collected in heparinized test tubes and were processed for analysis within 12 h. PBMC were isolated by Ficoll-Hypaque (Amersham Biosciences-Uppsala, Sweden) density gradient centrifugation. PBMC were washed twice with phosphate-buffered saline (PBS) containing 0.1% sodium azide (Wako, Japan) and 2% fetal calf serum (FCS).

Arterial cord blood samples (5–10 mL) from nine healthy neonates were obtained, with consent, from the clamped umbilical cord and mononuclear cells were isolated similarly.

FACS analysis

Cells were incubated with various monoclonal antibodies (mAbs) for 30 min at 4°C in the dark, washed and analyzed using a FACScan (Becton Dickinson) as previously described (12). The used mAbs included allophycocyanin (APC)-conjugated anti-CD3, phycoerythrin (PE)-conjugated anti-CD19, fluorescein isothiocyanate (FITC)-conjugated anti-CD56, FITC or PE-conjugated anti-CD4, FITC-conjugated anti-CD8, PE-conjugated anti-CD25, FITC-conjugated anti-T-cell receptor (TCR)-αβ, PE-conjugated anti-TCR-γδ, biotin-conjugated anti-Vδ1, FITC-conjugated anti-Vδ2, PE-conjugated anti-V-α24, FITC-conjugated anti-V-β11 and PE-conjugated isotype control IgG1 (mouse IgG1). All mAbs, as well as Streptavidin-PE were purchased from Becton Dickinson Pharmingen (San Diego, CA) or Coulter Immunotec (Marseille, France).

Statistical analysis

In this study, coefficient of variation was used to determine the assay reproducibility. To evaluate the assay reproducibility, FACS analyses of phenotypes of PBMC from 6 normal health adults were performed two times using the same volume of blood obtained in different time. The coefficient of variation was <10% (range: 1.8–6.3%).

All the data were presented by mean ± SD. The comparisons among the Gr-vol, Gr-IS and Gr-tol were done by one-way analysis of variance (ANOVA) and Bonferroni test. Values of p < 0.05 was regarded as significant. StatView-J5.0 (SAS Institute, Cary, NC, USA) software on Windows XP was used for statistical calculations.

Results

Characteristics of patients

The characteristics of patients are summarized in Table 1. Blood samples were collected from LDLT patients and volunteers from October 2002 to April 2004 with the informed consent. Of the 12 patients in Gr-tol, 9 (9/12) were weaned completely from IS according to the Kyoto elective weaning protocol (10), while the other 3 patients (3/12) had discontinued IS for clinical reasons, including non-compliance of medication, PTLD and infection. The diagnoses before Tx in LDLT patients included biliary atresia (8/12 in Gr-tol and 13/19 in Gr-IS), Alagille syndrome, cryptogenic liver cirrhosis, propionic acidemia and so on. Most patients (10/12 in Gr-tol and 18/19 in Gr-IS) received ABO-identical or compatible grafts. Routine blood tests demonstrated that the total leukocyte, ALT, AST, T-Bil and CRP were comparable among Gr-tol, Gr-IS and Gr-vol. (mean ± SD) (Tables 1 and 2).

Table 1.  The profiles of patients
 SexPrimary diseasesAgeYear of TxWean IS yearABO matchingALTASTT-BilCRP
  1. Patients' age, gender, primary disease, years after liver transplantation (LTx), and years of weaning off immunosuppression (IS) are indicated. Whether IS was electively weaned by protocol (P) or by clinical reasons (NP) is also indicated. If NP, reasons for cessation of IS are shown together. NP+, PTLD (post-transplant lymphoproliferative disorder); NP++, cholangitis and renal disorder; NP+++, diarrhea; BA, biliary artersia; HCC, hepatocellular carcinoma.

Gr-tol
1NP+MCryptogenic liver cirrhosis1485Compatible857110
2NP++FBA21125Identical31260.90
3NP+++FBA443Incompatible27430.50.3
4PFBA11104Identical14230.80.6
5PFIntrahepatic cholestatic15125Compatible56520.20
6PFPropionyl acidemia631Incompatible16350.80
7PMAlagille syndrome1592Identical45410.90
8PFBA874Identical14340.60
9PFBA1196Identical19190.30.2
10PFBA991Identical13260.80
11PFBA1193Identical33310.60
12PFBA23113Identical21200.90
Gr-IS
1MBA54 Identical14350.50.3
2FBA136 Identical951132.40.1
3MBA167 Identical40341.30.1
4FBA172 Identical25290.90
5MCongenital liver fibrosis132 Compatible9230.30
6FBA128 Identical24380.60
7MIntrahepatic cholestasis249 Compatible1726910
8MAlagille Syndrome, HCC61 Identical51610.90.1
9MHepatic veno-occlusive disease54 Compatible34350.40
10FBA85 Compatible36541.10
11MBA, Polysplenia syndrome43 Incompatible23471.20.1
12FBA53 Identical16300.40.4
13FBA64 Identical19310.60.1
14MCryptogenic liver cirrhosis115 Compatible38391.60.1
15MBA98 Identical14270.90
16FBA87 Identical23300.50
17FBA84 Identical20340.90.1
18FBA76 Identical25340.50.5
19FBA66 Identical14330.50.5
Summary
Gr-tol
n = 12F (n = 10)
M (n = 2)
 12 ± 69 ± 34 ± 2 31 ± 2135 ± 150.7 ± 0.30.1 ± 0.2
Gr-IS
n = 19F (n = 10)
M (n = 9)
 10 ± 55 ± 2  36 ± 3842 ± 200.9 ± 0.50.1 ± 0.2
Gr-vol
n = 24F (n = 10)
M (n = 14)
Mild cardiac diseases11 ± 5 19 ± 924 ± 120.7 ± 0.10.1 ± 0.1
Table 2.  The absolute number of PBMC subsets in the peripheral blood
 Gr-volGr-ISGr-tolGr-tol vs. Gr-volGr-IS vs. Gr-volGr-tol vs. Gr-IS
  1. NS: p > 0.2.

WBC (×109/L)6.0 ± 1.85.6 ± 1.36.0 ± 2.5NSNSNS
Lymphocytes (/μL)2052 ± 6602211 ± 7471956 ± 714NSNSNS
T cells (/μL)1260 ± 5631479 ± 4821267 ± 503NSNSNS
B cells (/μL)272 ± 123313 ± 150366 ± 177NSNSNS
NK cells (/μL)315 ± 158306 ± 249180 ± 175NSNSNS
NKT cells (/μL)0.6 ± 0.70.2 ± 0.40.2 ± 0.1p = 0.1p < 0.05NS
CD4+ T cells (/μL)696 ± 330786 ± 286673 ± 293NSNSNS
CD8+ T cells (/μL)403 ± 249530 ± 204449 ± 212NSNSNS
CD4+CD25+ cells (/μL)118 ± 43110 ± 64118 ± 56NSNSNS
CD4+CD25high+ cells (/μL)38 ± 1721 ± 1644 ± 17NSp < 0.01p < 0.01
TCR αβ cells (/μL)1175 ± 5481335 ± 4631159 ± 470NSNSNS
TCR γδ cells (/μL)124 ± 85130 ± 7792 ± 46NSNSNS
Vδ1 γδ T cells (/μL)15 ± 1138 ± 2747 ± 33p < 0.01p < 0.05NS
Vδ2 γδ T cells (/μL)93 ± 8067 ± 5333 ± 11p < 0.05NSNS

Alterations in T cells, B cells and NK cells

The percentage of total T cells (CD3+) did not differ among the three groups (Gr-tol, Gr-IS and Gr-vol; 66 ± 13%, 68 ± 11% and 60 ± 11%, NS, respectively) (Figure 1A). In contrast, the percentage of B cells (CD19+) was higher in Gr-tol than those in Gr-IS and Gr-vol (Gr-tol, Gr-IS and Gr-vol; 19 ± 4%, 14 ± 6% and 12 ± 5%: Gr-tol vs. Gr-IS; p = 0.1: Gr-tol vs. Gr-vol; p < 0.05) (Figure 1B). There was no difference in the percentage of CD4+ T cells among the three groups (Gr-tol, Gr-IS and Gr-vol; 53 ± 7%, 53 ± 7% and 56 ± 12%, NS, respectively) (Figure 1C). The percentage of CD8+ T cells did not differ among the three groups, either (Gr-tol, Gr-IS and Gr-vol; 35 ± 6%, 36 ± 7% and 31 ± 11%, NS, respectively) (Figure 1D). The percentage of NK cells (CD3CD56+) was decreased in Gr-tol, compared with those in Gr-IS and Gr-vol (Gr-tol, Gr-IS and Gr-vol; 8 ± 6%, 13 ± 7% and 15 ± 5%: Gr-tol vs. Gr-IS; p = 0.1: Gr-tol vs. Gr-vol; p < 0.05) (Figure 1E). With respect to the absolute number of each subset, there was no significant difference among the three groups (Table 2).

Figure 1.

Analyses for T cells, CD4+, CD8+, B cells and NK cells in age-matched volunteers (Gr-vol), LDLT patients on IS (Gr-IS) and operationally tolerant patients (Gr-tol). A minimum of 5000 cells was acquired in the lymphocyte gate for each analysis. There was no significant difference among Gr-tol, Gr-IS and Gr-vol in the percentage of T cells, CD4+ and CD8+ T cells (T cells, CD4+ and CD8+ T cells; 66 ± 13%, 53 ± 7% and 35 ± 6% in Gr-tol; 68 ± 11%, 53 ± 7% and 36 ± 7% in Gr-IS; 60 ± 11%, 56 ± 12% and 31 ± 11% in Gr-vol, NS, respectively) (A, C, D). Gr-tol demonstrated an increase in the percentage of B cells, compared with those in Gr-IS and Gr-tol (Gr-tol, Gr-IS and Gr-vol; 19 ± 4%, 14 ± 6% and 12 ± 5%: Gr-tol vs. Gr-IS; p = 0.1: Gr-tol vs. Gr-vol; p < 0.05) (B). In Gr-tol, a decrease was found in the percentage of NK cells, compared with those in Gr-IS and Gr-tol (Gr-tol, Gr-IS and Gr-vol; 8 ± 6%, 13 ± 7% and 15 ± 5%: Gr-tol vs. Gr-vol; p < 0.05: Gr-tol vs. Gr-IS; p = 0.1) (E).

Decreased NKT cells (Vα24+Vβ11+)

NKT cells in Gr-vol samples comprised only 0.06 ± 0.06% of the CD3+ T cells and were significantly reduced in Gr-tol and Gr-IS patients (Gr-tol and Gr-IS; 0.02 ± 0.01% and 0.01 ± 0.03%, vs. Gr-vol; p < 0.05 and p < 0.01, respectively) (Figure 2A, B). The absolute number of NKT cells also tended to be decreased both in Gr-tol and Gr-IS, compared with that in Gr-vol (Gr-tol, Gr-IS and Gr-vol; 0.2 ± 0.1/μL, 0.2 ± 0.4/μL and 0.6 ± 0.7/μL: Gr-tol vs. Gr-vol; p = 0.1: Gr-IS vs. Gr-vol; p < 0.05) (Table 2).

Figure 2.

Analysis for NKT cells (CD3+ gated Vα24+Vβ11+) in age-matched volunteers (Gr-vol), LDLT patients on IS (Gr-IS) and operationally tolerant patients (Gr-tol). 200 000 of T cells (CD3+) were acquired for each analysis. The representative FACS profiles of NKT cells in each group are shown (A). The frequency of NKT cells was significantly less both in Gr-tol and Gr-IS groups than that in Gr-vol (Gr-vol, Gr-IS and Gr-tol; 0.06 ± 0.06%, 0.01 ± 0.03% and 0.02 ± 0.01%: Gr-tol vs. Gr-vol; p < 0.05: Gr-IS vs. Gr-vol; p < 0.01, respectively) (B).

Increased CD4+CD25high+ cells

CD4+CD25+ T cells present in PBMC displayed a range of intensities of CD25+ expression from intermediate to high levels, whereas in the cord blood, the majority of CD4+CD25+ cells exhibited high CD25 intensity as shown in Figure 3A and in agreement with a previous report (13). The 9 different samples of cord blood analyzed showed a similar tendency (data not shown). It has been proposed that CD4+CD25high+ cells represent Treg and CD4+CD25low+ cells activated/memory-type cells (13). Based on these results, CD4+CD25high+ cells were defined as a fluorescence intensity of CD25+ over 102 in our study. The percentage of CD4+CD25high+ cells was significantly higher in Gr-tol, compared with those in the other two groups (Gr-tol, Gr-IS and Gr-vol; 2.3 ± 0.6%, 0.9 ± 0.7%, and 1.8 ± 0.6%: Gr-tol vs. Gr-IS; p < 0.01: Gr-tol vs. Gr-vol; p < 0.05). The percentage of CD4+CD25high+ cells in Gr-IS was even lower than that in Gr-tol. (Figure 3D) (Gr-IS vs. Gr-vol; p < 0.01). On the other hand, there was no significant difference in the percentage of total CD4+CD25+ cells among the three groups (Gr-tol, Gr-IS and Gr-vol; 6.1 ± 2.0%, 5.2 ± 3.2% and 5.8 ± 1.8%, NS, respectively) (Figure 3C). The absolute number of CD4+CD25high+ cells was significantly increased in Gr-tol as compared with that found in Gr-IS sample. However, this did not differ between Gr-tol and Gr-vol. (Gr-tol, Gr-IS and Gr-vol; 44 ± 17/μL, 21 ± 16/μL and 38 ± 17/μL: Gr-tol vs. Gr-IS; p < 0.01: Gr-tol vs. Gr-vol; NS) (Table 2). Similarly to the percentage, unlike CD4+CD25high+ cells, there was no difference in the absolute number of total CD4+CD25+ cells among the three groups (Gr-tol, Gr-IS and Gr-vol; 118 ± 56/μL, 110 ± 64/μL and 118 ± 43/μL, NS, respectively) (Table 2).

Figure 3.

Analyses for CD4+CD25+ cells in age-matched volunteers (Gr-vol), LDLT patients on IS (Gr-IS) and operationally tolerant patients (Gr-tol). The representative FACS profiles of CD4+CD25high+ are shown. 15 000 of CD4+CD25+ cells were acquired in the lymphocyte gate for each analysis. Analysis using nine cord blood displayed that CD25 level of CD25high+ cells mostly is above 102 (A). Based on the analysis of cord blood, the percentage of CD4+CD25high+ cells was examined in Gr-vol, Gr-IS and Gr-tol. Figure 3B showed the typical FACS profile of the percentage of CD4+CD25high+ cells in Gr-tol and Gr–vol. Gr-tol demonstrated an increase in the percentage of CD4+CD25high+ cells (Gr-tol, Gr-IS and Gr-vol; 2.3 ± 0.6%, 0.9 ± 0.7% and 1.8 ± 0.6%: Gr-tol vs. Gr-IS; p < 0.01: Gr-tol vs. Gr-vol; p < 0.05) (D). However, no significant difference was found in the percentage of total CD4+CD25+ cells among three groups (Gr-tol, Gr-IS and Gr-vol; 6.1 ± 2.0%, 5.2 ± 3.2% and 5.8 ± 1.8%, NS, respectively) (C).

Altered γδ T cells subset composition

Neither the proportions nor the absolute numbers of αβ and γδ T cells differed among the three groups (Gr-tol, Gr-IS and Gr-vol; αβ/γδ T cells, 91 ± 4% [1159 ± 470/μL]/8 ± 4% [92 ± 46/μL], 90 ± 6% [1335 ± 463/μL]/9 ± 5% [130 ± 77/μL] and 90 ± 6%[1175 ± 548/μL]/10 ± 6% [124 ± 85/μL], NS, respectively) (Figure 4A, B and Table 2). γδ T cells express one of the three different Vδ gene segments, Vδ1, Vδ2 or Vδ3, representing distinctive functional subsets (14). We examined Vδ1 and Vδ2 γδ T cells in peripheral blood. In Gr-vol, Vδ2 γδ T cells predominated and Vδ1 γδ T cells were rare (Figure 4C); consistent with a previous report (14). Thus, Vδ1/Vδ2 γδ T-cells ratio was less than 1.0 in most samples of Gr-vol. In marked contrast, Vδ1 γδ T cells rather than Vδ2 γδ T cells predominated in Gr-tol (Figure 4C). Conversely to Gr-vol, Vδ1/Vδ2 γδ T-cells ratio was over 1.0 in most samples of Gr-tol. In Gr-IS, Vδ1/Vδ2 γδ T-cells ratio was in the middle of those in Gr-tol and Gr-vol (Gr-tol, Gr-IS and Gr-vol; Vδ1/Vδ2 γδ T-cells ratio, 1.5, 0.8 and 0.3: Gr-tol vs. Gr-vol; p < 0.01: Gr-tol vs. Gr-IS; p < 0.05) (Figure 4D).

Figure 4.

Analyses for αβ/γδ T cells and Vδ1, Vδ2 γδ T cells in age-matched volunteers (Gr-vol), LDLT patients on IS (Gr-IS) and operationally tolerant patient (Gr-tol). 10 000 of T cells (CD3+) were acquired for analyses of αβ/γδ T cells. There was no significant difference among Gr-tol, Gr-IS and Gr-vol in the percentage of αβ and γδ T cells (Gr-tol, Gr-IS and Gr-vol; αβ T cells; 91 ± 4%, 90 ± 6% and 90 ± 6%, γδ T cells; 8 ± 4%, 9 ± 5% and 10 ± 6%, NS, respectively) (A, B). 200 000 of CD3+ lymphocytes were acquired for analyses of Vδ1 and Vδ2 γδ T cells. In Gr-vol, Vδ2 γδ T cells predominated while Vδ1 γδ T cells were rare. In quite a contrast, however, Vδ1 rather than Vδ2 predominated in Gr-tol (C). Thus, the conversed ratio of Vδ1/Vδ2 γδ T cells was observed in mostly Gr-tol patients. However, this was less frequently found in Gr-IS than in Gr-tol (Gr-tol, Gr-IS and Gr-vol; Vδ1/Vδ2 γδ T-cells ratio, 1.5, 0.8 and 0.3: Gr-tol vs. Gr-vol; p < 0.01: Gr-tol vs. Gr-IS, p < 0.05) (D).

An increase of Vδ1 γδ T cells and a decrease of Vδ2 γδ T cells in the percentage and the absolute number were only significant between Gr-tol and Gr-vol, but not between Gr-tol and Gr-IS (Gr-tol, Gr-IS and Gr-vol; the percentage of Vδ1/Vδ2 γδ T cells, 3.7 ± 2.4%/2.9 ± 1.2%, 2.7 ± 1.9%/4.8 ± 3.8% and 1.3 ± 0.8%/7.1 ± 5.9%: Gr-tol vs. Gr-vol; p < 0.01/p < 0.05, Gr-tol vs. Gr-IS; NS/NS, respectively) (Gr-tol, Gr-IS and Gr-vol; the absolute number of Vδ1/Vδ2 γδ T cells, 47 ± 33/μL/33 ± 11/μL, 38 ± 27/μL/67 ± 53/μL and 15 ± 11/μL/93 ± 80/μL: Gr-tol vs. Gr-vol; p < 0.01/p < 0.05: Gr-tol vs. Gr-IS, NS/NS, respectively) (Table 2).

Discussion

We have demonstrated that successful achievement of a complete IS-free state (operational tolerance) after LTx is feasible in a substantial proportion (approximately 40%) of LDLT patients by an elective weaning protocol (10). Nonetheless, some patients (approximately 25%) encountered rejection while undergoing weaning from IS. It is, therefore, essential to identify and characterize the immunological differences that will enable patients in these two distinct populations to be distinguished reliably.

In the present study, we analyzed systemically the phenotypes of lymphocyte subsets in PBMC derived from operationally tolerant patients (completely IS free) after LDLT and compared them with those of PBMC from LDLT patients on IS and age-matched volunteers. Several alterations in the lymphocyte subsets were revealed in the operationally tolerant patients (Gr-tol). These included an increase in the percentage of CD4+CD25high+ cells, B cells and Vδ1/Vδ2 γδ T-cells ratio and a decrease of the percentage of NK cells.

CD4+CD25high+ Treg is reported to play a pivotal role in establishment of tolerance in various experimental Tx models (5). There are reports, which showed the increase in Treg in operationally tolerant patients or clinically stable renal Tx patients (15,16). The findings in our study were consistent with these experiments and clinical reports (Figure 3B, D). Functional analyses of the CD4+CD25high+ cells present in the peripheral blood of operationally tolerant LDLT patients are now in progress to investigate the possibility that they have regulatory activities.

A subset of γδ T cells, sharing a similar phenotype with IEL, has been shown to function directly as regulatory cells in the maintenance of tolerance to rat liver allografts (7). In humans, however, the role of γδ T cells in clinical organ transplantation remains elusive. In reviewing literatures, γδ T cells could be involved in human renal allograft rejection while γδ T cells also may participate in human cardiac allograft acceptance (17,18). Our present results show that Vδ1 γδ T cells, which are normally found in the epithelial tissues, rather than Vδ2 γδ T cells predominate in the PBMC of operationally tolerant LDLT patients (Figure 4C). It is reported that Vδ1 γδ T cells become a dominating population in the peripheral blood lymphocytes during normal, but not abortive pregnancy (19). Furthermore, Vδ1 γδ T cells in the deciduas during early pregnancy were reported to produce Th2-type cytokines such as IL-10 and TGF-β, suggesting the possible involvement of Vδ1 γδ T cells in maintaining the maternal tolerance to the fetus (20). These results raise the possibility that the increased number of Vδ1 γδ T cells present in the PBMC of tolerant LDLT recipients may play a role in the establishment of operational tolerance developed to the liver graft.

In contrast, NK cells were significantly reduced in the PBMC of operationally tolerant patients and NKT cells decreased in LDLT patients either in operational-tolerance group or IS group. This could be due either to absolute reduction of these cells or to their recruitment to the tissues including the liver graft. NKT cells uniformly decreased after LDLT. Therefore, a decrease of NKT cells does not seem to be specific to operational tolerance in LDLT. However, NKT cells were found to accmulate within liver and have been implicated to be involved in its acceptance in rodent models (8). Thus, further analysis of these two lymphocyte subsets in the hepatic graft is essential for the evaluation of their roles.

An unexpected finding in the present study was an increase in the number of B cells in LDLT recipient exhibiting operational tolerance. Very recently, however, the same phenomenon has been demonstrated in operational tolerance after human renal Tx (16). It is not clear whether operational tolerance necessarily protects against development of chronic rejection to which humoral response to donor-antigen may contribute. Therefore, we are currently investigating histological findings of accepted graft by performing needle biopsy.

In this study, the average time (±SD) after LDLT and weaning of IS were 9 (±3) and 4 (±2) years in Gr-tol, respectively. However, it is still unidentified when these lymphocyte phenotypes changes occurred and how they contributed to immune regulation according to different period after LDLT. Further functional assay and prospective longitudinal studies for PBMC and investigation of liver biopsy may offer us better understanding of operating mechanisms in tolerance.

In conclusion, we have shown intriguing differences in the phenotype of lymphocytes present in PBMC in operationally tolerant patients and age-matched volunteers with normal liver function by FACS analysis. The possible functional involvement of these lymphocyte subsets in the maintenance of tolerance remains to be analyzed carefully. Meanwhile, present results may provide a significant clue to select the Tx patients for the safe and successful weaning from IS.

Acknowledgments

We are grateful to Professor Koizumi Akiko at Kyoto University School of Public Health for his suggestions on statistics methods. We thank Ms. J. Sano and N. Nagaosa for expert technical assistance, Ms. N. Yoshida and K. Fukui for preparing the manuscript, and especially Dr. H. Doi at the Department of Pediatric for collecting blood samples.

Presented in part at the American Transplant Congress 2004 May 14–19, 2004, Boston, USA and winner of a Young Investigator Award and at the XX International Congress of the Transplantation Society 2004, September 5–10, 2004, Vienna, Austria.

KJW holds a Royal Society Wolfson Merit Award.

Ancillary