Experimental Model Of Autoimmune Hepatitis Research Articles

Bifidobacterium mitigates autoimmune hepatitis by regulating IL-33-induced Treg/Th17 imbalance via the TLR2/4 signaling pathway.

The present work aims to evaluate the efficacy of Live Combined Bifidobacterium, Lactobacillus and Enterococcus Capsules (LCBLECs), a probiotic drug containing Bifidobacterium, in the treatment of autoimmune hepatitis (AIH). In this study, a mouse model of experimental autoimmune hepatitis (EAH) was established to investigate the effects of LCBLECs on AIH. The results showed that LCBLECs improved dysbiosis of gut microbiota, reduced liver injury, restored liver function, and maintained Treg/Th17 balance in EAH mice. In addition, LCBLECs restored Treg/Th17 balance in EAH mice by downregulating IL-33 production. Besides, LCBLECs also suppress IL-33 upregulation in EAH mice by inhibiting the TLR2/4 signaling pathway. Furthermore, LCBLECs also mitigated dysbiosis of gut microbiota and enhanced the efficacy of conventional treatment for AIH patients. To sum up, our findings revealed that LCBLECs exerted therapeutic effects on EAH mice by improving Treg/Th17 imbalance in an IL-33-dependent manner via the TLR2/4 signaling pathway and relieved the clinical symptoms of AIH patients, indicating Bifidobacterium supplementation with LCBLECs might be a potential adjuvant therapy for AIH treatment.

Lactobacillus improves the effects of prednisone on autoimmune hepatitis via gut microbiota-mediated follicular helper T cells

BackgroundAutoimmune hepatitis (AIH) is a chronic, immune-mediated liver dysfunction. The gut microbiota and T follicular helper (Tfh) cells play critical roles in the immunopathogenesis and progression of AIH. We aimed to investigate the effect of gut microbiota combined with prednisone therapy on Tfh cell response in AIH.MethodsSamples from AIH patients and mouse model of experimental autoimmune hepatitis (EAH) were analyzed using real-time quantitative polymerase chain reaction, enzyme-linked immunosorbent assay, western blotting, flow cytometry, and hematoxylin–eosin staining to determine the role of gut microbiota on AIH.ResultsLactobacillus significantly increased the levels of Bacteroides fragilis, Clostridium, Clostridium leptum, Bifidobacterium, and Lactobacillus and significantly enhanced the suppressive effects of prednisone on the levels of AIH clinical indexes in AIH patients. Lactobacillus exerts the same prptective effects as prednisone in EAH mice and enhanced the effects of prednisone. Lactobacillus also reinforced the inhibitory effects of prednisone on the levels of serum IL-21 and the proportions of Tfh cells in peripheral blood mononuclear cells. Mechanistically, prednisone and Lactobacillus regulated Tfh cell response in EAH mice in an MyD88/NF-κB pathway-dependent manner.ConclusionOur results suggested a therapeutic potential of Lactobacillus in the prednisone-combined treatment of AIH.6BN4VU1A1WC6Nqr62iBRGoVideo

Formononetin protects against concanavalin-A-induced autoimmune hepatitis in mice through its anti-apoptotic and anti-inflammatory properties.

Autoimmune hepatitis (AIH) is a chronic inflammatory liver disease that seriously threatens the health of humans globally. Formononetin (FMN) is a natural herb extract with multiple biological functions. In this study, an experimental model of AIH was established in mice through the use of concanavalin A (ConA). To investigate the effects of FMN on ConA-induced hepatitis, the mice were pretreated with 50 or 100 mg/kg body mass of FMN. The results show that FMN alleviated ConA-induced liver injury of mice in a dose-dependent manner. Moreover, pretreatment with FMN inhibited the apoptosis of hepatocytes in the ConA-treated mice through downregulating the expression of pro-apoptotic proteins (Bax, cleaved caspase 9, and cleaved caspase 3) and upregulating the expression of anti-apoptotic protein (Bcl-2). It was also found that the levels of proinflammatory cytokines were greatly reduced in the serum and liver tissues of mice pretreated with FMN. Further studies showed that FMN reduced the level of phosphorylated nuclear factor kappa B (p-NF-κB) p65 and enhanced the level of IκBα (inhibitor of NF-κB), suggesting that FMN inhibits the activation of the NF-κB signaling pathway. In addition, FMN inhibited activation of the NOD-like receptor protein 3 (NLRP3) inflammasome. Therefore, FMN could be a promising agent for the treatment of AIH.

Exploration the significance of Tfh and related molecules on C57BL/6 mice model of experimental autoimmune hepatitis.

This study aimed at establishing the autoimmune hepatitis (AIH) model of C57BL/6 mice, and examining the expression and significance of T follicular helper (Tfh) cells, T follicular regulatory (Tfr) cells, effector B cells and other indicators in this experimental autoimmune hepatitis (EAH) model. C57BL/6 mice in experimental group were administered by intraperitoneal injection after fully emulsified on 1st day and 7th day with 0.5mL of 0.5-2.0g/L S-100 and an equal volume of Buddha Complete Adjuvant (CFA). The levels of serum alanine transaminase (ALT), aspartate aminotransferase (AST), and interleukin-21 (IL-21) were tested by the corresponding kit. Tfr, Tfh and B cell subsets were quantified by flow cytometry. Histological pathology was completed by pathological section experiments. In comparison with that in the healthy controls (HC), significantly increased numbers of serum ALT and AST, Tfr, IL-21 was observed. The hepatic lobules and hepatocyte cords were severely disorganized. The results of liver pathological changes and serum index changes were similar to the chronic and progressive pathogenesis and pathology of AIH patients, providing a detailed pathological basis for the basic research and clinical experiments of AIH.

The Imbalance between Foxp3+Tregs and Th1/Th17/Th22 Cells in Patients with Newly Diagnosed Autoimmune Hepatitis

This study is aimed at examining the potential role of regulatory T- (Treg-) Th1-Th17-Th22 cells in the pathogenic process of autoimmune hepatitis (AIH). The numbers of Foxp3+Tregs and Th1, Th17, and Th22 cells were measured in 32 AIH patients using flow cytometry. Moreover, a murine model of experimental autoimmune hepatitis (EAH) was also established and used to investigate the function of Treg-Th1-Th17-Th22 cells in disease progression. AIH patients undergoing an active state had significantly decreased numbers of CD3+CD4+CD25+Foxp3+Tregs and increased numbers of CD3+CD4+CD25−Foxp3+T, CD3+CD4+IFN-γ+Th1, CD3+CD4+IL-17+Th17, and CD3+CD4+IL-2+Th22 cells as well as higher levels of Th1/Th17/Th22-type cytokines compared to AIH patients in remission and HC. Additionally, the numbers of CD3+CD4+CD25+Foxp3+Tregs were negatively correlated with the numbers of Th1-Th17-Th22 cells. Also, the serum levels of IL-17A and IL-22 were correlated positively with liver injury (ALT/AST), whereas the serum levels of IL-10 were correlated negatively with hypergammaglobulinaemia (IgG, IgM) in AIH patients. Interestingly, the percentages of spleen Tregs, expression of Foxp3 mRNA, and liver IL-10 levels decreased, whereas the percentages of spleen Th1-Th17-Th22 cells, expression of T-bet/AHR/RORγt mRNA, and liver IFN-γ, IL-17, and IL-22 levels increased in the murine model of EAH. Our findings demonstrated that an imbalance between Tregs and Th1-Th17-Th22 cells might contribute to the pathogenic process of AIH.

BMSCs-derived miR-223-containing exosomes contribute to liver protection in experimental autoimmune hepatitis

Autoimmune hepatitis is a chronic inflammatory disease in the liver with potential to the development of liver fibrosis. Recent evidences suggest that bone marrow derived mesenchymal stem cells (BMSCs) may exert its therapeutic activity through exosomes. Moreover, miR-223 is highly expressed in BMSCs and plays an important role in autoimmune diseases. Therefore, in this study, hepatoprotective role of BMSCs and miR-223 was investigated in both mice and hepatocytes. Liver antigen S100 was used to establish autoimmune hepatitis model in mice while LPS and ATP were used to establish cell injury model in hepatocyte. Before the experiments, BMSCs were infected with pre-miR-223 and transfected with miR-223 inhibitor respectively. Exosomes from bone marrow stem cells were isolated by ultracentrifugation. Liver injury was evaluated by serum levels of ALT and AST as well as liver histology. Inflammation and cell death were examined by inflammatory cytokines and lactase dehydrogenase respectively. Both BMSCs-exo and BMSCs-exomiR−223(+) significantly reversed either S100 or LPS/ATP induced injury in mice and hepatocytes. Meanwhile, the expressions of cytokines, NLRP3 and caspase-1 were also downregulated by BMSCs-exo and BMSCs-exomiR−223(+) at both protein and mRNA levels in mice and hepatocytes. Moreover, BMSCs-exomiR−223(−) reverses the effects of BMSCs-exo and BMSCs-exomiR−223(+) in mouse AIH and in hepatocytes. In conclusion, bone marrow stem cell derived exosomes can protect liver injury in an experimental model of autoimmune hepatitis and the mechanism could be related to exosomal miR-223 regulation of NLRP3 and caspase-1.

Celecoxib attenuates depressive-like behavior associated with immunological liver injury in C57BL/6 mice through TNF-α and NF-κb dependent mechanisms

AimDepression associating patients with chronic liver diseases is a major treatment goal. This study aimed to evaluate the potential hepatoprotective and antidepressant effects of celecoxib in a model of experimental autoimmune hepatitis (EAH) and depressive-like behavior in C57BL/6 mice. Main methodsEAH was induced by immunization with S-100 liver antigen emulsified in complete Freund's adjuvant (CFA). Mice were randomly allocated to 5 groups; control phosphate buffered saline group; control CFA group; EAH group, and 2 groups of EAH plus celecoxib (7.5 or 15mg/kg/d respectively). Mice were assessed behaviorally by novelty-suppressed test, tail suspension test, locomotor assessment and forced swimming tests. Serum liver enzymes and hepatic hydroxyproline content were biochemically analyzed. Histopathological analysis for liver and brain sections and immunohistochemical studies for hepatic and hippocampal tumor necrosis factor (TNF-α), nuclear factor Kappa-B (NF-κB) and caspase-3 were performed. Key findingsEAH group exhibited significant depressive-like changes, increase in liver enzymes and hepatic hydroxyproline content. Signs of autoimmune hepatitis and structural changes in hippocampus were confirmed by histopathological studies. Immunohistochemical examination revealed overexpression of hepatic and hippocampal TNF-α, NF-κB and caspase-3 positive cells. Celecoxib (7.5mg/kg/d) significantly ameliorated hepatic biochemical changes, hepatic and hippocampal histopathological and immunohistochemical changes induced in EAH group. Celecoxib (15mg/kg/d) significantly ameliorated the behavioral changes, histopathological and immunohistochemical changes in hippocampus, with non-significant change in hepatic biochemical profile, histopathological and immunohistochemical changes induced in EAH group. SignificanceThe celecoxib (7.5mg/kg/d) through its anti-inflammatory effect may represent a new therapeutic approach to treat autoimmune hepatitis associated with depressive symptoms.

Depletion of B cells induces remission of autoimmune hepatitis in mice through reduced antigen presentation and help to T cells

Autoimmune hepatitis (AIH) is known as a T cell-mediated disease. However, AIH patients refractory to conventional treatment have been successfully treated with anti-CD20-mediated B-cell depletion. The aim of this project was to understand the immunological changes underlying the AIH remission caused by B-cell depletion in an experimental model of AIH. C57BL/6 AIH mice, xenoimmunized with DNA coding for human liver antigens, were treated with a single dose of depleting mouse anti-CD20 antibody at the peak of liver inflammation. Liver inflammation, alanine aminotransferase levels, chemokine (C-X-C) ligand 10 expression, and circulating B-cell, autoantibody, and total immunoglobulin G levels were monitored following depletion. T-cell and B-cell phenotype and function were characterized. Administration of a single dose of anti-CD20 resulted in a drastic reduction of liver inflammation accompanied by a significant reduction of alanine aminotransferase levels and of proinflammatory chemokine (C-X-C) ligand 10 expression. The treatment did not result in significant changes in total immunoglobulin G levels or autoantibodies. There were significantly more naive and less antigen-experienced CD4+ and CD8+ T cells, and T-cell proliferation was significantly reduced following anti-CD20 treatment. B cells served as antigen-presenting cells to CD4+ T cells. Anti-CD20 treatment also led to a profound reduction of T follicular helper cells. B cells play an active role in the pathogenesis of AIH in antigen presentation processes and the modulation of T-cell functions and influence the T follicular helper-cell population; this active role of B cells could explain the success of B-cell depletion for remission of AIH despite its classification as a T cell-mediated autoimmune liver disease.

Immunomodulation of liver injury by Ascaris suum extract in an experimental model of autoimmune hepatitis.

Adult worm extract from Ascaris suum (Asc) has immunosuppressive activity and elicits Th2/IL-4/IL-10 response. This study evaluated the prophylactic and therapeutic effect of Asc in a murine model of concanavalin A (ConA)-induced autoimmune hepatitis (AIH). BALB/c mice received ConA, iv, (20 mg/kg), and three groups of animals were formed: (1) AIH, received only ConA; (2) AIH + Asc prophylactic, treated with Asc (1 mg/ml), ip, 30 min before of the AIH; and (3) AIH + Asc therapeutic, treated with Asc 2 h after the AIH. Plasma transaminase and immunoglobulins (measured at 8 and 24 h and 7 days after treatment) and cytokine production (IL-4, IL-10, IL-13, and IFN-γ) by splenocytes upon ConA and Asc stimulus were compared. The livers were weighed and examined histologically. In the AIH group, there was an increase in liver weight, transaminase levels, and total immunoglobulins. These parameters were reduced by 8-24 h and 7 days in the prophylactic group, but in the therapeutic group, only on day 7. The survival rate of mice in the AIH group was 38.5%, compared to 67% in the therapeutic Asc group. The survival rate of the animals with AIH that were prophylactically treated with Asc was 100%. A decrease of cellular infiltration and high levels of IL-4, IL-10, and IL-13 were induced by Asc. An increase of liver fibrosis was also observed, but with less intensity with prophylactic treatment. Thus, the Ascaris components have an inhibitory effect on AIH, with an intense Th2 immune response.

IL-17 contributes to autoimmune hepatitis

The role of interleukin-17 (IL-17) in autoimmune hepatitis (AIH) was investigated. A mouse model of experimental autoimmune hepatitis was established, and the syngeneic S-100 antigen emulsified in complete Freud's adjuvant was injected intraperitoneally into adult male C57BL/6 mice. The IL-17 expression in serum and the livers of the mice models was detected by using ELISA and immunohistochemistry, respectively. IL-17 neutralizing antibody was used to study the biological effect of IL-17 in the experimental AIH. IL-17 neutralizing antibody in vivo administration alleviated the hepatic inflammation and ALT level in the AIH model. IL-17 in the peripheral blood mononuclear cells (PBMC) of AIH patients was measured by using real-time PCR method. The results showed that IL-17 level was significantly up-regulated in AIH patients and mice models. It was concluded that IL-17 contributed to the development of AIH and might be a potential therapeutic target of AIH.

Role of 2B4‐mediated signals in the pathogenesis of a murine hepatitis model independent of Fas and Vα14 NKT cells

Concanavalin A (Con A)-induced hepatitis is a T-cell-mediated murine experimental model of autoimmune hepatitis. Mice lacking Valpha14 NKT cells were found to be less sensitive to this hepatitis and the MRL/Mp-Fas(lpr/lpr) (MRL/lpr; i.e. Fas deficient) mice were also less sensitive. We report herein that MRL/Mp-Fas(lpr/lpr)-Sap(rpl/-) (MRL/lpr/rpl) mice lack Valpha14 NKT cells and are deficient in the Fas antigen but sensitive to Con A-induced hepatitis. The signaling lymphocytic activation molecule (SLAM)-associated protein (SAP) is an adaptor molecule containing a Src homology 2 (SH2) domain. We previously reported new mutant mice found among MRL/lpr mice and revealed that SAP deficiency led to the regression of autoimmune phenotypes in mutant MRL/lpr/rpl mice. It was also revealed that CD4(+) and CD8(+) T cells were effector cells and that blockade of 2B4, one of the SLAM family receptors, inhibited the induction of hepatitis in MRL/lpr/rpl mice. These data suggest that signals mediated by molecules other than SAP from 2B4 in T cells played important roles in the induction of hepatitis in MRL/lpr/rpl mice.

788 Development of a New Mouse Model of Fatal Autoimmune Hepatitis

Background: An aberrant regulation of autoreactive T cells against liver antigens appears to initiate development of autoimmune hepatitis (AIH), characterized by mononuclear-cell infiltration and autoantibody production. Because of the lack of animal models developing spontaneous autoimmune liver disease resembling AIH, molecular mechanisms involved in development of AIH are still unclear. Although Con A-induced acute hepatic injury is considered to be an experimental model of AIH, Con A-induced acute hepatic injury does not produce circulating autoAbs and a single intravenous injection of Con A in mice induces rapid injury of hepatocytes with a striking increase in plasma transaminase levels within 24 h. Thus, this model is not likely to provide the clues for resolving molecular mechanisms of how an aberrant regulation of autoreactive T cells against liver antigens is initiated In Vivo. Aim: The aim of this study is to develop a new mouse model of spontaneous AIH and to examine molecular mechanisms involved in development of AIH. Methods: Using a negative co-stimulatory molecule, programmed cell death 1, deficient mice (PD-1 KO mice), we performed depletion of naturally arising CD4+CD25+ regulatory T cells (Treg cells). After Treg-cell depletion In Vivo, we monitored histological findings of the liver and production of autoantibody, and performed immunohistological analysis, flow cytometry examination and adoptive transfer experiments. Results: Neither Treg-cell depleted wild-type mice nor PD1 KO mice showed inflammation of the liver. However, age-matched Treg-cell depleted PD1 KO (Treg-dep-PD-1 KO mice) produced anti-hepatocyte antibodies and developed fatal hepatitis. The fatal hepatitis showed severe CD4+ and CD8+ T-cell infiltration invading parenchyma and massive lobular necrosis without bile duct destruction and fibrosis. Transfer of total splenocytes from Treg-dep-PD-1 KOmice into immunodeficient host, RAG2 deficient mice developed hepatitis. However, transfer of CD4+ T-cell-depleted splenocytes into RAG2 deficient recipient mice did not induce any inflammation of the liver. These data indicate that hepatitis in Treg-dep-PD-1 KO mice is a bona fide T cell-mediated autoimmune disease and that CD4+ T cells plays an essential role in the induction of AIH. Conclusion: This study indicates that Treg-dep-PD-1 KO mice are the first mouse model of spontaneous fatal AIH, and that immune dysregulation by concurrent loss of Treg cells and PD-1-mediated signaling can trigger activation of autoreactive T cells to induce fatal AIH.

Pivotal role of Smad3 in a mouse model of T cell-mediated hepatitis

Transforming growth factor beta (TGFbeta) promotes hepatocellular apoptosis and suppresses hepatic lymphocyte responses in part through activation of Smad3. The purpose of the current study was to determine the importance of Smad3 signaling in an experimental model of autoimmune hepatitis induced by concanavalin A (ConA), a process involving T cell activation and hepatocellular apoptosis. C57Bl/6 wild-type (Wt) or Smad3-deficient (Smad3(-/-)) mice were injected intravenously with 15 mg/kg ConA or vehicle. Nine hours post ConA injection, Wt mice presented with severe hepatitis as assessed by increased liver transferases. This injury was associated with eosinophil accumulation and preceded at 3 hours post-injection by significant increases in hepatic T helper 1 (interferon gamma) and T helper 2 (interleukin-4) cytokine production. Absence of Smad3 significantly blunted hepatocellular injury 9 hours post ConA injection, which was associated with reduced early T helper 1 and T helper 2 cytokine production and eosinophil accumulation. Smad3(-/-) livers also showed significant reductions in hepatocellular apoptosis as assessed by terminal UTP nick-end labeling when compared to ConA-treated Wt mice in conjunction with reduced caspase 3 cleavage, which was likely mediated by a Smad3-dependent inhibition of the survival factor extracellular signal-regulated kinase 1/2. In vitro, Smad3(-/-) hepatocytes were resistant to TGFbeta-induced apoptosis, and this protection was dependent on extracellular signal-regulated kinase activation. Together, these results show, for the first time, the significance of Smad3 signaling in autoimmune hepatitis, underlining the control of Smad3-dependent TGFbeta signaling on proinflammatory cytokine production, eosinophil recruitment, and hepatocellular apoptosis. Interruption of this pathway could be beneficial clinically to limit acute fulminant liver pathologies.

Pivotal role of Smad3 in T cell‐mediated hepatitis

Transforming growth factor β promotes hepatocellular apoptosis and suppresses hepatic lymphocyte responses in part through activation of Smad3. The purpose of the current study was to determine the importance of Smad3 signaling in an experimental model of autoimmune hepatitis induced by Concanavalin A (ConA), a process involving T cell activation and hepatocellular apoptosis. C57Bl/6 wild type (wt) or Smad3-deficient (Smad3−/−) mice were injected i.v. with 15mg/kg ConA or vehicle. Nine hours post-ConA injection, wt mice presented with severe hepatitis measured by liver transaminases in conjunction with significant increases in hepatic T helper 1 (Th1; interferon gamma) and Th2 (interleukin 4) cytokine production. Absence of Smad3 significantly blunted hepatocellular injury 9 hours post-ConA injection despite similar increases in Th1 and exaggerated Th2 cytokine production. Furthermore, in vitro T cell activation assays also revealed an imbalanced Th1:Th2 cytokine production in Smad3−/− lymphocytes when compared to wt controls. Finally, Smad3−/− livers showed significant reductions in hepatocellular apoptosis as assessed by terminal UTP nick end labeling when compared to ConA-treated wt mice. Together, these studies demonstrate the multifunctional importance of Smad3 signaling in the activation of T lymphocytes and induction of hepatocellular apoptosis in an experimental model of autoimmune hepatitis. This work was supported by NIH grant AA014243.

Suppression of murine experimental autoimmune hepatitis by T-cell vaccination or immunosuppression.

Patients with autoimmune hepatitis (AIH) usually require immunosuppressive therapy for many years, if not for a lifetime. Experimental immunotherapy such as T-cell vaccination aims at manipulating the immune system in such a way that autoimmunity is specifically regulated to enable long-lasting correction of the disease process. We aimed to test the feasibility of T-cell vaccination as well as conventional immunosuppression in the murine model of experimental autoimmune hepatitis (EAH). EAH was induced in 5- to 7-week-old BALB/c mice by immunization with syngeneic liver homogenate in complete Freund's adjuvant. For T-cell vaccination, splenocytes were removed from animals 14 days after induction of EAH and from control animals, and activated in vitro by mitogen stimulation with Concanavalin A (Con A). Activated T cells were irradiated and injected at 5 x 10(7) cells per animal as T-cell vaccine. Immunosuppression in control animals was performed with prednisolone with or without azathioprine. T-cell vaccination with T cells from EAH animals, but not with irrelevant T cells, was able to protect animals from EAH, reducing the average disease severity from 2.2 (+/-0.3) to 0.5 (+/-0.3) (P < .01). T-cell vaccination was also able to treat EAH, because application of the vaccine 2 weeks after induction of the disease significantly reduced disease activity at week 4 from 2.4 (+/-0.4) to 1.1 (+/-0.2) (P < .05). Both passive transfer of disease and the capacity to protect by T-cell vaccination was mediated by CD4 T cells. Specific cellular recognition of activated disease-inducing T cells could be detected in vaccinated animals. Immunosuppressive drugs could also suppress EAH. Thus, T-cell vaccination in EAH is feasible and effective. Stimulation of a regulatory T-cell network is the likely mechanism of action by which T-cell vaccination can suppress EAH.

Cell-mediated cytotoxicity of sensitized spleen cells against target liver cells--in vivo and in vitro study with a mouse model of experimental autoimmune hepatitis.

Spleen cells obtained from C57BL/6 (B6) mice with an experimental autoimmune hepatitis were transferred to normal C57BL/6 recipient mice. Most prominent liver damages occurred in the recipient mice injected with sensitized nylon wool column-adherent spleen cells from the donor mice. Production of such liver damage was blocked by treatment of the sensitized adherent spleen cells with anti-Thy 1,2 monoclonal antibody and complement before injection. Based on these in vivo results, a microcytotoxicity assay was performed using isolated C57BL/6 hepatocytes as target cells and sensitized spleen cells obtained from hepatitis donor mice as effector cells. The fraction of sensitized nylon wool-adherent spleen cells demonstrated a high cytotoxic activity against isolated syngeneic hepatocytes, although the other fractions and spleen cells of control animals showed no such effect. The cytotoxic activity of sensitized-adherent spleen cells against target hepatocytes was significantly reduced after treatment with anti-Thy 1,2 antibody and complement, but it increased after depletion of B cells and Fc receptor-bearing T-cells. Although these sensitized nylon wool-adherent spleen cells showed high cytotoxic activities against syngeneic hepatocytes, their cytotoxicity against allogeneic hepatocytes was lower. They exerted no cytotoxic activity against syngeneic renal glomerular cells and EL-4 thymoma cells. These results suggest that sensitized T-cells in the nylon wool column-adherent fraction play the role of cytotoxic killer cells against target liver cells in vitro.