Selective reduction of natural killer T cells in the bone marrow of aplastic anaemia

T cell-mediated suppression of haematopoiesis is believed to play an important role in the pathophysiology of aplastic anaemia (AA) and in the pancytopenia of some myelodysplastic syndromes (MDS). Natural-killer T (NKT) cells belong to a unique lymphocyte subset that expresses an invariant T-cell receptor (TCR), consisting of Valpha24JalphaQ, and common NK cell surface markers. NKT cells have been hypothesized to play a role in immune regulation, and many human autoimmune conditions are associated with NKT cell deficiency. Here we investigate the role of NKT cells in AA and MDS patients. Flow cytometry demonstrated that NKT cells, unlike other T-lymphocyte subpopulations, were disproportionally decreased in AA and MDS marrow. When we compared variability within the CDR3 region of Valpha24 in CD4-CD8- T cells derived from AA and healthy individuals, the CDR3 size of Valpha24 cells showed a polyclonal distribution in AA patients, while in control subjects a typical oligoclonal or monoclonal pattern was found. Southern blot and sequence analysis of Valpha24 polymerase chain reaction products revealed that the NKT cell-specific JalphaQ region was predominant in control subjects, whereas it was not, or only very weakly, detected in AA and MDS patients. These results show that NKT cells are profoundly decreased in AA and MDS, and their deficiency may, as in other human autoimmune diseases, play a role in the local immune dysregulation in AA and MDS.     

Dendritic cells are targets for human invariant Valpha24+ natural killer T-cell cytotoxic activity: an important immune regulatory function

OBJECTIVE: Human invariant Valpha24+ natural killer T (NKT) cells, a subpopulation of NK cell-receptor (NKR-P1A)-expressing T cells with an invariant Valpha24JalphaQ T-cell receptor (TCR), are stimulated by the glycolipid a-galactosylceramide (KRN7000), in a CD1d-dependent, TCR-mediated fashion. Little is known about invariant Valpha24+ NKT cell function or mechanisms of effector activity. Evidence suggests this cell population protects against autoimmunity and has antitumor effects against leukemia and solid tumors. MATERIALS AND METHODS: We compared the phenotype and function of invariant Valpha24+ NKT cells, from patients with chronic myeloid leukemia (CML) and normal donors, generated by stimulation of peripheral blood mononuclear cells with alpha-galactosylceramide pulsed monocyte-derived dendritic cells. The CD4(-)CD8(-) (double negative) population was studied further. RESULTS: Activated human invariant Valpha24+ NKT cells were cytotoxic against autologous and allogeneic peripheral blood dendritic cells and monocyte-derived dendritic cells but not against autologous or allogeneic T-cell PHA blasts, B-cell lymphoblastoid cell lines, monocytes, or leukemic cells from patients with CML. The findings are consistent with previous observations showing the importance of CD1d in target cell recognition. None of the Valpha24+ NKT cell lines expressed the NK markers CD16, CD56, CD94, or killer inhibitory receptors, but all expressed NKR-P1A. There was no difference in phenotype, function, or ease of generation of invariant Valpha24+ NKT cells between normal donors and patients with CML. CONCLUSION: Based on our results and the previous evidence linking reduced Valpha24+ NKT cells to autoimmunity, we propose that double-negative Valpha24+ NKT cells have important immune regulatory functions, including contribution to the prevention of excessive antigen stimulation by virtue of cytotoxic activity against antigen presenting cells, particularly in dendritic cells.     

Valpha24+ natural killer T-cell responses against T-acute lymphoblastic leukaemia cells: implications for immunotherapy

Human Valpha24+ natural killer T (NKT) cells correspond to mouse Valpha14+ NKT cells, both cell types use an invariant T-cell receptor-alpha chain and are activated by glycolipids in a CD1d-dependent manner. Mouse Valpha14+ NKT cells have been reported to have an antitumour effect in vivo. Human Valpha24+ NKT cells can kill a proportion of tumour cells in a CD1d-dependent manner in vitro. We report here that many human leukaemic T-cell lines express CD1d and can be directly killed by Valpha24+ NKT cells. This killing activity was enhanced in the presence of alpha-galactosylceramide (alpha-GalCer), a ligand of Valpha24+ NKT cells. Moreover, primary leukaemic T cells from five of eight T-cell acute lymphoblastic leukaemia (T-ALL) patients expressed CD1d and were good targets of Valpha24+ NKT cells. This cytotoxicity was increased in the presence of alpha-GalCer. Our results suggest that T-ALL is a good candidate for Valpha24+ NKT-cell-based immuno-cell therapy.     

Atherosclerotic abdominal aortic aneurysm and the interaction between autologous human plaque-derived vascular smooth muscle cells, type 1 NKT, and helper T cells

Immune cell infiltration, vascular smooth muscle cell (VSMC) proliferation, and apoptosis are pathological hallmarks of atherosclerosis. The multifocal, chronic, and inflammatory nature of this disease of the cardiovascular system complicates targeted cellular therapy and emphasizes the need to understand the role and interaction of immune cells with VSMCs. We characterized the immune cell subsets present in human atherosclerotic tissue derived from atherosclerotic abdominal aortic aneurysm (AAA) and expanded them to study their interaction with autologous plaque-derived VSMCs in vitro. We show here that apart from T lymphocytes, plaque infiltrates consist of lots of NK cells and significant proportions of NKT cells that express T cell receptor (TCR) alphabeta, CD4, and the NK markers CD56 and CD161. The infiltrates are predominantly IFN-gamma-producing Type 1 lymphoid cells. When cocultured, the T and NKT cells adhere to VSMCs. CD4+ T cells enhance VSMC proliferation. VSMCs in turn enhance CD4+CD161+ NKT but not CD4+ or CD8+ T cell proliferation. CD4+CD161+ NKT cells inhibit VSMC proliferation by inducing apoptosis. Our results suggest that the interactions of Type 1 CD4+ T and CD4+CD161+ NKT cells with VSMCs may regulate VSMC proliferation and death respectively in atherosclerosis and the balance of these interactions could determine plaque stability.     

Recovery of Valpha24+ NKT cells after hematopoietic stem cell transplantation

Human Valpha24+ natural killer T (NKT) cells have an invariant T-cell receptor-alpha chain and are activated in a CD1d-restricted manner. Valpha24+ NKT cells are thought to regulate immune responses and to play important roles in the induction of allograft tolerance. In this report, we analyzed the recovery of Valpha24+ NKT cells after hematopoietic stem cell transplantation and its correlation with graft-versus-host disease (GVHD). Patients who received a dose-reduced conditioning regimen, antithymocyte globulin- or CAMPATH-1H-containing conditioning regimen were excluded. NKT cells were reconstituted within 1 month after transplantation in peripheral blood stem cell transplantation recipients, while their numbers remained low for more than 1 year in bone marrow transplantation (BMT) recipients. The number of Valpha24+ NKT cells in BMT recipients with acute GVHD was lower than that in patients without acute GVHD, and both the CD4+ and CD4- Valpha24+ NKT subsets were significantly reduced. With regard to chronic GVHD, BMT recipients with extensive GVHD had significantly fewer Valpha24+ NKT cells than other patients. Furthermore, the number of CD4+ Valpha24+ NKT cells was also significantly reduced in patients with chronic extensive GVHD. Our results raise the possibility that the number of Valpha24+ NKT cells could be related to the development of GVHD.     

Changes in immune cell frequencies after cyclophosphamide or mycophenolate mofetil treatments in patients with systemic lupus erythematosus

This study was designed to explore the profile of immune cell subsets, including T, B, natural killer (NK), and NKT cells, in systemic lupus erythematosus (SLE) patients, and to determine their relationships with the clinical index and the effects of cyclophosphamide (CYC) and mycophenolate mofetil (MMF) treatment. SLE patients (n?=?28) and age/sex-matched healthy controls (n?=?28) were evaluated. The patients were equally divided into two treatment groups: intravenous drip (IVD) with CYC and prednisolone, and oral MMF and IVD with prednisolone. SLE peripheral blood samples were taken immediately prior to treatment and after 4?weeks of drug treatment. T, B, NK, and NKT cell subsets were measured by flow cytometry. Double-stranded DNA antibody and Sm antibody were detected by indirect immunofluorescence. Serum C3, C4, and C-reactive protein were determined by scatter turbidimetry. The percentages of CD3+CD4+ T, CD3-CD16CD56+ NK, and CD3+CD16CD56+ NKT cells and the CD4+/CD8+ ratio were significantly lower in SLE patients, while CD3+CD8+ T and CD3-CD19+ B cells were higher than the controls. The lymphocyte subsets were significantly correlated with the SLE disease activity index (SLEDAI) and complement factors (C3, C4). Four weeks of CYC or MMF treatment led to a significant increase in CD3+CD4+ T cells (P?相似文献   

Changes in natural killer T cells subsets during therapy in type C hepatitis and hepatocellular carcinoma.

Natural killer T (NKT) cells share features of both classical T cells and NK cells. NKT are heterogenous populations, and recognize glycolipids associated with CD1d molecule. We investigated Th1/Th2 cytokine production as well as frequency and phenotype of circulating NKT cells in 14 healthy subjects and in patients during therapy with type C chronic hepatitis (CH; 14 cases) and hepatocellular carcinoma (HCC; 13 cases). Peripheral blood mononuclear cells (PBMC) were obtained before and 2 weeks later interferon (IFN)/ribavirin and radiofrequency ablation therapy for CH and HCC, respectively. PBMC were cultured for 10 days with alpha-galactosylceramide (alpha-GalCer) and interleukin-2 (IL-2). Frequencies and IFN-gamma/IL-4 production of NKT cells were analyzed using flow cytometry. Intrahepatic lymphocytes were analyzed in seven CH patients with liver biopsy specimen. Prevalence of circulating Valpha24+CD3+ T cells was 0.9+/-0.9% of PBMC for controls and increased to 8.5+/-8.9% (p<0.001) in response to alpha-GalCel. Similar frequency and expansion were noted in CH. The frequency increased during therapy. The prevalence in HCC tended to be high compared to controls and response to alpha-GalCel was well. Although frequency of Valpha24+Vbeta11+CD3+ T cells was low in all groups, the distribution pattern was similar to Valpha24+Vbeta11-CD3+ T cells. Prevalence of CD56+CD3+ T cells was low independent of therapy in CH (2-3%) compared to 5.0+/-4.0% of controls, although response to alpha-GalCel was not impaired. IFN-gamma production of Valpha24+CD3+ T cells did not differ among groups, but became greater after treatment in contrast to lowered IL-4 production. Frequencies of NKT populations were higher in liver than in peripheral blood. Our study suggests that CD1d-reactive T cells have distinct distribution in different populations and therapy for patients alters cytokine response of NKT cells.     

Expansion of pulmonary CD8+CD56+ natural killer T-cells in hypersensitivity pneumonitis

BACKGROUND: Natural killer T (NKT) cells, a newly identified subgroup of T cells with immunoregulatory function, may be implicated in the pathogenesis of interstitial lung disease (ILD). METHODS: We used multiparameter flow cytometry with antibodies to CD3, CD4, CD8, CD14, CD19, CD45, CD16/56, CD56, CD161, and Valpha24 invariant T-cell receptor (TCR) in BAL fluid (BALF) to examine the frequency and distribution of pulmonary NKT cells in several cases of ILD. We included 57 patients with sarcoidosis and 17 patients with hypersensitivity pneumonitis. RESULTS: We found significantly higher frequencies of pulmonary NKT cells in patients with hypersensitivity pneumonitis in comparison to the other study patients with ILD (median proportion of NKT cells, 11%; range, 3 to 38%; vs 3%; range, 0 to 16%; p < 0.0001). In contrast, there was no difference in the proportion of conventional natural killer cells. We found that a major subset of NKT cells in the BALF of patients with hypersensitivity pneumonitis was a CD8+CD56+ population that did not express the invariant TCR. CONCLUSIONS: These results suggest the involvement of NKT cells in the pathogenesis of hypersensitivity pneumonitis.     

人骨髓间充质干细胞对间质性肺纤维化抗损伤治疗研究

目的 研究骨髓间充质干细胞在间质性肺纤维化中的抗损伤治疗作用机制.方法 通过体外诱导的自然杀伤T细咆(NKT细胞)模拟间质性肺纤维化炎症损伤特点.以干扰素γ(IFN-γ),CD3,白介素2刺激培养外周血单个核细胞,得到含高比例CD3+ CD56+ NKT细胞,将这种培养的CD3+ CD56+ NKT细胞与人骨髓间充质干...     

中国乙型肝炎患者外周血淋巴细胞亚群频率参考值范围

目的 调查中国乙型肝炎患者外周血淋巴细胞亚群频率参考值范围.方法 利用流式细胞术检测2846例乙型肝炎患者和117例健康人群外周血淋巴细胞亚群数值,调查我国健康人群和乙型肝炎人群的参考值范围.结果 调查了16～60岁健康人群和HBV感染相关的急性肝炎、慢性肝炎、重型肝炎和肝硬化人群外周血CD3+T淋巴细胞、CD3+CD...     

Molecular cytogenetics of stem cells: target cells of chromosome aberrations as revealed by the application of fluorescence in situ hybridization to fluorescence-activated cell sorting

The lineage involvement in stem cell disorders, such as chronic myeloid leukemia (CML) and myelodysplastic syndrome (MDS), remains unclear. To explore this issue, we used fluorescence in situ hybridization for cells sorted by fluorescence-activated cell sorting (FACS) from 12 patients with chronic-phase CML. Philadelphia chromosome (Ph) was found in pluripotent stem cells (CD34+Thy-1+), B cells (CD34+CD19+), and T/natural killer (NK) progenitor cells (CD34+CD7+) collected by FACS from bone marrow cells. B (CD19+), T (CD3+), and NK (CD3-CD56+) cells showed a marked decrease in Ph+ cells between progenitor cells and mature cells The Ph+ T and NK cells decreased to below background levels. These data suggest that Ph+ lymphocytes either do not differentiate or are eliminated during their maturation process Among 7 MDS patients associated with trisomy 8, sorted lymphocytes from peripheral blood did not have +8. CD34+ subpopulations from bone marrow including B,T/NK progenitors, and pluripotent progenitor cells also did not have +8.Trisomy 8 was identified from the level of multipotent colony-forming units (CD34+CD33+), and the lymphoid lineage was not involved. Thus, MDS with trisomy 8 conceivably arises from nonlymphoid progenitor cells, sparing T, B, or NK cells. Further studies using molecular cytogenetics may clarify the mechanism of leukemia happening at the level of stem cells.     

Expansion of CD1d-restricted NKT cells in patients with primary HIV-1 infection treated with interleukin-2

Innate CD1d-restricted natural killer T (NKT) cells are infected and lost in HIV-1-infected patients, and this could contribute to HIV-1 pathogenesis because NKT cells play an important role in directing both adaptive and innate immunity. Administration of interleukin-2 (IL-2) to HIV-1-infected patients leads to substantial and sustained CD4+ T-cell expansion, involving both naive and memory cells. We investigated whether IL-2 treatment could restore the NKT cell compartment in patients with primary HIV-1 infection. We show that IL-2 combined with effective antiretroviral therapy (ART) resulted in significant expansion of CD1d-restricted NKT cells. Expansion occurred in both the CD4- and CD4+ subsets of NKT cells, and expanded cells expressed the CD161 maturation marker while expression of the HIV coreceptor CCR5 was reduced. These data indicate that IL-2 treatment in combination with effective ART is beneficial for the restoration of innate NKT cell immunity in patients with primary HIV-1 infection.     

In vitro differentiation of natural killer T cells from human cord blood CD34+ cells

Natural killer T (NKT) cells are involved in innate immune defence and also in the regulation of adaptive immune responses. However, the development of NKT cells in vitro has not been fully characterized and culture conditions have not been fully optimized. In the present study, we found that an NKT cell fraction developed during the in vitro culture of cord blood (CB) CD34+ cells, and this was subsequently characterized both phenotypically and morphologically. CD34+ cells purified from 10 human CB were cultured in the presence of several cytokines and analysed by flow cytometry, light microscopy and electron microscopy. The NKT cell fraction, defined phenotypically (CD3+CD16+CD56+CD94+) as expressing the invariant T-cell receptor Valpha24 and Vbeta11, appeared in the CD56hi fractions. Intracytoplasmic staining demonstrated that interferon-gamma and interleukin 4 (IL-4) were detected in the CD56hi fractions. IL-15 was essential and, in combination with either flt3-ligand (FL) or stem cell factor (SCF), was sufficient to induce the development of NKT cells. The phenotype of the NKT cell fraction was CD45RO+CD45RA- and CD4+CD8alpha+. Morphologically, they were very large, with either round or oval nuclei, moderately condensed chromatins, voluminous weakly basophilic cytoplasm and various cytoplasmic granules such as dense core granules, multivesicular bodies, and intermediate form granules. When CD34+ cells purified from bone marrow (BM) were compared with those from CB, the latter were consistently more efficient at generating CD56hi NKT cell fractions. In conclusion, IL-15 in combination with FL and/or SCF can induce the differentiation of NKT cells from human CB CD34+ cells.     

Glycolipid activation of invariant T cell receptor+ NK T cells is sufficient to induce airway hyperreactivity independent of conventional CD4+ T cells

Asthma is an inflammatory lung disease, in which conventional CD4+ T cells producing IL-4/IL-13 appear to play an obligatory pathogenic role. Here we show, in a mouse model of asthma, that activation of pulmonary IL-4/IL-13 producing invariant TCR+ CD1d-restricted natural killer T (NKT) cells is sufficient for the development of airway hyperreactivity (AHR), a cardinal feature of asthma, in the absence of conventional CD4+ T cells and adaptive immunity. Respiratory administration of glycolipid antigens that specifically activate NKT cells (alpha-GalactosylCeramide and a Sphingomonas bacterial glycolipid) rapidly induced AHR and inflammation typically associated with protein allergen administration. Na?ve MHC class II-deficient mice, which lack conventional CD4+ T but have NKT cells, showed exaggerated baseline AHR and, when challenged with alpha-GalactosylCeramide, demonstrated even greater AHR. These studies demonstrate an expanded role for NKT cells, in which NKT cells not only produce cytokines that influence adaptive immunity but also function as critical effector cells that can induce AHR. These results suggest that NKT cells responding to glycolipid antigens, as well as conventional CD4+ T cells responding to peptide antigens, may be synergistic in the induction of AHR, although in some cases, each may independently induce AHR.     

Quantitation and phenotypic analysis of natural killer T cells in primary biliary cirrhosis using a human CD1d tetramer

BACKGROUND & AIMS: Natural killer T (NKT) cells are a subset of lymphocytes incriminated in playing an important role in the modulation of the innate immune response and the development of autoimmunity. However, there have been only limited studies attempting to quantitate the number of NKT cells in autoimmune disease, particularly because of difficulties associated with definition of this subpopulation. METHODS: We used a human CD1d (hCD1d) tetramer produced by a baculovirus expressing recombinant CD1d protein complexed with alpha-galactosylceramide (alpha-GalCer) and quantitated hCD1d tetramer reactive cells in blood and liver from controls and patients with primary biliary cirrhosis (PBC). RESULTS: The majority of CD1d-alphaGalCer-restricted NKT cells were positive for TCR Valpha24 and Vbeta11. There was a distinct CD4- CD8+ population within the CD1d-alphaGalCer-restricted NKT cells in addition to the CD4- CD8- and CD4+ CD8- population. The frequency of CD1d-alphaGalCer-restricted NKT cells was similar between blood and liver in healthy individuals. In contrast, the frequency of CD1d-alphaGalCer-restricted NKT cells in the liver was significantly higher than in the blood of PBC patients. The frequency of CD1d-alpha-GalCer-restricted NKT cells in the liver was also significantly higher in PBC patients than in healthy individuals. CONCLUSIONS: The frequency and function of such cells should be studied not only in blood but also in the target organ of the autoimmune disease. Selective enrichment of CD1d-alphaGalCer-restricted NKT cells at the site of inflammation is observed in PBC, suggesting a role of these cells in the development of PBC.     

Dysfunction of T cell receptor AV24AJ18+, BV11+ double-negative regulatory natural killer T cells in autoimmune diseases

OBJECTIVE: We examined the reduction of T cell receptor (TCR) AV24+,BV11+ CD4-,CD8- (double-negative [DN]) natural killer T (NKT) cells in peripheral blood lymphocytes (PBLs) from patients with rheumatoid arthritis (RA), systemic lupus erythematosus (SLE), systemic sclerosis (SSc), and Sjogren's syndrome (SS) to analyze why NKT cells are selectively reduced in autoimmune diseases, and to examine whether nonresponse to alpha-galactosylceramide (alpha-GalCer) is due to an abnormality in the antigen-presenting cells (APCs) or NKT cells. METHODS: Peripheral blood from patients with RA (n = 20), SLE (n = 18), SSc (n = 13), and SS (n = 17), as well as from healthy donors (n = 13) and patients with Beh?et's disease (BD; n = 20), was examined by flow cytometry to determine the number of TCR AV24+,BV11+ DN T cells. PBLs from 10 RA, 10 SLE, 8 SSc, and 9 SS patients, as well as from 7 healthy subjects, were cultured in vitro with alpha-GalCer, and the number of TCR AV24+,BV11+ DN NKT cells was estimated. APCs from responder and nonresponder patients were cocultured with NKT cells from responder and nonresponder patients. RESULTS: The mean +/- SEM number of TCR AV24+,BV11+ DN NKT cells per ml of whole blood was found to be 48.8+/-10.0 in RA patients, 50.6+/-12.9 in SLE patients, 80.8+/-30.6 in SSc patients, and 40.0+/-11.7 in SS patients, while 290.0+/-69.6 and 321.2+/-103.4 NKT cells were present in healthy subjects and BD patients, respectively (P < 0.01). Three of 10 RA patients, 5 of 10 SLE patients, 4 of 8 SSc patients, and 6 of 9 SS patients (a total of 18 of 37 patients, or 48.6%) responded to alpha-GalCer, indicating that patients could be divided into two groups: alpha-GalCer responders and nonresponders. In contrast, NKT cells from all healthy subjects proliferated against alpha-GalCer. APCs from all nonresponder patients were found to function as alpha-GalCer-presenting cells, while NKT cells from nonresponders did not expand even in the presence of APCs from normal responders. CONCLUSION: These findings strongly suggest that patients with autoimmune diseases can be divided into two groups (alpha-GalCer responders and nonresponders). They also suggest that the reduced numbers of NKT cells in patients with autoimmune diseases may be due to an inadequate amount of alpha-GalCer-like natural ligands (i.e., adequate in only 48.6% of patients) for the induction of NKT cells in vivo, or to a dysfunction in the NKT cells themselves (in 51.4% of patients).     

T细胞、NKT和NK细胞在结核病诊断中的意义探讨

目的探讨CD3+T细胞、CD3+CD16+CD56+N KT细胞及CD3-CD16+CD56+N K细胞在肺结核患者外周血的表达状态及其临床意义。方法采用流式细胞仪分析123例肺结核患者(73例初治肺结核和50例复治肺结核)及83例健康者(43例结核菌素皮试阳性和40例结核菌素皮试阴性)外周血中T细胞、NKT和NK细胞的表达率。结果初、复治肺结核患者的NKT细胞表达率明显高于健康者(P0.01),但初治肺结核患者的NK细胞、T细胞的表达率与健康者相比差异无统计学意义(P0.05)。T细胞表达率在不同程度的初治肺结核患者中差异有统计学意义(P0.01),疾病越重,表达越高。NKT、NK细胞差异无统计学意义(P0.05)。复治肺结核患者的T细胞表达率显著高于初治肺结核患者(P0.01),但NK细胞、NKT细胞的表达率在2组间差异无统计学意义(P0.05)。结论T细胞、NKT细胞是结核病细胞免疫的重要组成部分。对NKT细胞只在活动性肺结核患者才会呈现高表达,测定外周血NKT细胞可能成为辅助诊断活动性肺结核的手段之一。     

Recent advances in the role of NKT cells in allergic diseases and asthma

Asthma is the result of chronic airway inflammation that is dominated by the presence of eosinophils and CD4⁺ T lymphocytes. CD4⁺ T cells include several subsets and play a critical role in orchestrating the inflammation, predominantly by secreting interleukin-4 and interleukin-13. Recently, research identified a new subset of T cells, natural killer T (NKT) cells, which also express the CD4 marker. In contrast to conventional CD4⁺ T cells, NKT cells do not respond to peptide antigens, but rather to glycolipids. In animal models of asthma, direct activation of NKT cells by glycolipids results in the secretion of extensive amounts of cytokines and triggers the development of airway hyperreactivity. Moreover, in patients with chronic asthma, NKT cells can be found in bronchoalveolar lavage fluids in significant amounts. These data strongly suggest that NKT cells play an important role in asthma pathogenesis.     

Wall shear stress and intrahepatic leukocytes of graft in living related donor liver transplantation

BACKGROUND/AIMS: We investigated the influence of HTK solution against natural killer T cells and thymic T cells in liver graft before and after perfusion in adult living related donor liver transplantation. METHODOLOGY: Graft samples were obtained before liver resection, after perfusion, and one hour after liver transplantation. Flowcytometry analysis was conducted using several human natural killer markers; CD16, CD56, CD57, and CD161. RESULTS: Natural killer T cells existed prominently in the liver leukocytes compared with their presence in peripheral blood lymphocytes, and the difference was significant. CD56+ T and CD161+ T cells, in comparison with CD16+ T cells and CD57+ T cells, were especially numerous in the liver. The proportion of CD56+ T and CD161+ T cells increased in the graft immediately after perfusion with HTK solution. However, CD16+ T cells and CD57+ T cells decreased in the graft immediately after perfusion and reperfusion of portal blood flow. Thymus-derived cells also decreased significantly after perfusion. The proportion of CD56+ T cells among CD3+ cells showed a significant increase immediately after perfusion. All types of natural killer cells in the graft immediately increased after perfusion by HTK solution and reperfusion of portal blood flow. Compared with CD57+ NKT cells, CD56+ NKT cells showed a significant tendency to stay in the liver graft against the perfusion. CD57+ NKT cells tended to wash out from the liver into the systemic circulation. Moreover, thymus-derived T cells showed the strongest tendency to wash out from the liver graft. CONCLUSIONS: CD56+ NKT cells and natural killer cells are more involved in local immunity, whereas thymus-derived cells and CD57+ NKT cells are involved in regulation of systemic immunity. Alloimmunity between local and systemic systems may be affected by the dynamic changes in hepatic circulation associated with living related donor liver transplantation.