Liver-infiltrating Lymphocytes Research Articles

Single-Cell Heterogeneity of the Liver-Infiltrating Lymphocytes in Individuals with Chronic Echinococcus multilocularis Infection.

Human alveolar echinococcosis (AE) is a tumor-like disease predominantly located in the liver. The cellular composition and heterogeneity of the lesion-infiltrating lymphocytes which produce an "immunosuppressive" microenvironment are poorly understood. Here, we profiled 83,921 CD45+ lymphocytes isolated from the peripheral blood (PB), perilesion (PL), and adjacent normal (AN) liver tissue of four advanced-stage AE patients using single-cell RNA and T-cell receptor (TCR) sequencing technology. We identified 23 large clusters, and the distributions and transcriptomes of these cell clusters in the liver and periphery were different. The cellular proportions of exhausted CD8+ T cells and group 2 innate lymphoid cells (ILC2s) were notably higher in PL tissue, and the expression features of these cell subsets were related to neoplasm metastasis and immune response suppression. In the 5 CD8+ T-cell populations, only CD8+ mucosa-associated invariant T (MAIT) cells were enriched in PL samples and the TRAV1-2_TRAJ33_TRAC TCR was clonally expanded. In the 11 subsets of CD4+ T cells, Th17 cells and induced regulatory T cells (iTregs) were preferentially enriched in PL samples, and their highly expressed genes were related to cell invasion, tumor metastasis, and inhibition of the inflammatory immune response. Exhaustion-specific genes (TIGIT, PD-1, and CTLA4) were upregulated in Tregs. Interestingly, there was a close contact between CD8+ T cells and iTregs or Th17 cells, especially for genes related to immunosuppression, such as PDCD1-FAM3C, which were highly expressed in PL tissue. This transcriptional data set provides valuable insights and a rich resource for deeply understanding the immune microenvironment in AE, which could provide potential target signatures for AE diagnosis and immunotherapies.

Interleukin-24 Limits Tumor-Infiltrating T Helper 17 Cell Response in Patients with Hepatitis B Virus-Related Hepatocellular Carcinoma.

Interleukin (IL)-24 is a multifunction cytokine in infectious diseases and cancers. IL-17-secreting CD4+ T (Th17) and CD8+ T (Tc17) cells promotes pathogenesis of hepatitis B virus (HBV)-associated liver disorders. However, the regulatory role of IL-24 to Th17/Tc17 cell response was not fully elucidated during HBV infection and HBV-hepatocellular carcinoma (HCC). In this study, plasma and peripheral blood mononuclear cells were isolated from 27 chronic hepatitis B (CHB) patients, 42 HBV-HCC patients and 17 normal controls (NC). Liver-infiltrating lymphocytes (LILs) were prepared from tumor and para-tumor tissues of 17 HBV-HCC patients, whereas CD4+ T cells in LILs were purified. LILs were stimulated with recombinant human IL-24. CD3+CD4+IL-17+ Th17 cells and CD3+CD8+IL-17+ Tc17 cells were investigated by flow cytometry. IL-24 level was measured by enzyme-linked immunosorbent assay. Purified CD4+ T cells were polarized for Th17 cells, and the regulatory role of IL-24 to liver-infiltrating Th17 polarization was assessed. There were no significant differences of peripheral Th17 or Tc17 cell percentage among NC, CHB, and HBV-HCC patients. Liver-infiltrating Th17 and Tc17 cell proportion was reduced in tumor tissues compared with para-tumor tissues. In contrast, plasma IL-24 level was increased in CHB and HBV-HCC patients. Exogenous IL-24 stimulation (10 or 100 ng/mL) in vitro downregulated of Th17 frequency and IL-17 secretion in LILs from both para-tumor and tumor tissues without affecting cellular proliferation or Tc17 percentage. Only 100 ng/mL of IL-24 inhibited tumor-infiltrating Th17 polarization, and this process was accompanied by suppression of nuclear factor-κB (NF-κB) and p38 mitogen-activated protein kinase phosphorylation. In conclusion, IL-24 might dampen Th17 cell response through NF-κB pathway in HBV-HCC tumor microenvironment. Elevated IL-24 might enhance anti-tumor immune response in HBV-HCC patients.

Clinical features and mechanism of liver injury in patients with mild or moderate coronavirus disease 2019.

Background and AimWe aimed to identify clinical features that suggest that coronavirus disease 2019 (COVID‐19) should be a differential diagnosis in patients presenting with a chief complaint of fever and abnormal liver function.MethodsWe retrospectively studied the presence or absence of abnormal liver function in 216 patients diagnosed with mild–moderate severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) infection between February and September 2020.ResultsAbnormal liver function was observed in 51 patients with mild–moderate COVID‐19. The median peak aspartate aminotransferase (AST), alanine aminotransferase (ALT), and lactate dehydrogenase (LDH) levels were 57.5, 75.5, and 332.5 U/L, respectively. The median number of days from symptom onset to peak AST, ALT, and LDH were 8.5, 9, and 8.5, respectively. The median peak LDH/AST ratio was 9.0. Low lymphocyte‐to‐white blood cell ratio and elevated LDH were found to be independent contributing factors for intensive care unit (ICU) admission on a multivariate analysis.ConclusionsAST‐predominant AST/ALT/LDH elevation peaking 8–9 days after symptom onset and not accompanied by elevated alkaline phosphatase or gamma‐glutamyl transferase may be a useful clinical feature for differentiating COVID‐19 from other diseases. Since the median LDH/AST ratio was 9.0, it seems that the abnormal liver function caused by SARS‐CoV‐2 is an indirect damage to liver cells due to elevated cytokine levels caused by liver‐infiltrating lymphocytes. SARS‐CoV‐2 infection should be considered in patients presenting with a chief complaint of fever and liver injury; those with a high lymphocyte‐to‐white blood cell ratio or and a high LDH/AST ratio may be admitted to the ICU.

TNF-Producing Th1 Cells Are Selectively Expanded in Liver Infiltrates of Patients with Autoimmune Hepatitis.

Autoimmune hepatitis (AIH) is a chronic inflammatory liver disease that is believed to be driven by a CD4+ T cell response to liver Ags. However, the pathogenic function of CD4+ effector T cells in AIH is not fully understood. To characterize liver-infiltrating lymphocytes in AIH, we determined the cytokine production of infiltrating cells obtained from biopsy material by quantitative RT-PCR and flow cytometry. A cytokine quantitiative RT-PCR array of AIH specimens revealed that TNF was the most strongly upregulated cytokine, as compared with control livers. To confirm this finding, we determined the frequencies of TNF-producing CD4+ T cells in peripheral blood and in liver biopsy specimens in comparison with those of CD4+ T cells producing IFN-γ or IL-17. In AIH, TNF-producing CD4+ T cells were significantly expanded, both in blood and liver, whereas IL-17-producing CD4+ T cells were not. However, the majority of the TNF-producing CD4+ T cells in AIH also produced IFN-γ, suggesting that TNF producers might represent a pathogenic activation state of Th1 cells. Ag-specific stimulation of PBMC from AIH patients with the AIH-associated autoantigen SEPSECS resulted in significant TNF production only in patients manifesting SLA/LP autoantibodies targeting SEPSEC but not in healthy individuals who do not manifest this reactivity. Taken together, our findings indicated that TNF-producing CD4+ T cells are expanded in AIH, both in blood and in liver. TNF-producing CD4+ T cells in AIH seem to be aberrantly activated Th1 cells. Our findings provide a rationale for therapeutic efforts using TNF blockade in AIH.

TCR Vβ Usage of Peripheral Blood and Liver Infiltrating Lymphocytes in Patients with Chronic Hepatitis B

Introduction. Chronic hepatitis B (CHB) is still a public health problem and its mechanism remains unclear. In this study, we detect the skewness of T cell receptor beta chain variable gene (TCR Vβ) in peripheral blood lymphocytes (PBL) and the liver infiltrating lymphocytes (LIL) of patients with CHB; and hope to provide information for further research on the pathogenic mechanism of CHB.Material and methods. Fifteen patients with CHB, ten healthy volunteers and three patients with liver cysts were recruited as the subjects. The usage of TCR Vβ of PBL and LIL were measured and compared; the associations of the TCR Vβ usage of PBL with some hematological indices, including human leukocyte antigen (HLA) alleles, percents of CD4+ and CD8+ T cells, sera levels of HBV-DNA and IFN-γ, were analyzed.Results. In PBL, Vβ12 and Vβ13.1 were the highest predominant usage genes which usage frequencies were all 46.7%; Vβ23 was the key limited usage gene (40.0%). In LIL, the mainly predominant and limited usage gene was Vβ13.1 (73.3%) and Vβ23 (46.7%), respectively. About half of the patients with CHB with HLA-DR9 or HLA-DR12 showed the predominant usage of Vβ5.2 or Vβ13.2. In patients with CHB, the percentage of CD4+ T cells was 33.41 ± 5.39 %, that of CD8+ T cells was 28.67 ± 6.77 %; the concentration of IFN-γ was 182.52 ± 44.16 pg/mL. Compared to the healthy controls, there were significant differences for these data (P < 0.05). Neither ALT nor HBV-DNA was relative to the usage of TCR Vβ.Conclusions. PBL and LIL share the common sknewness of TCR Vβ genes which probably relates to some hematological indices. However, the roles of such similarities and associations in the development of CHB need further study.

Liver-infiltrating CD8(+) lymphocytes as prognostic factor for tumour recurrence in hepatitis C virus-related hepatocellular carcinoma.

Chronic liver inflammation and immune/inflammatory response promote hepatocellular carcinoma. The aim of this study was to characterize the immune status of HCV-related cirrhosis in patients with hepatocellular carcinoma (HCV-HCC) as compared to HCV patients without hepatocellular carcinoma. Immune markers (CD3, CD4, CD8, CD20, CD56, TCRγδ, FoxP3) and gene expression profiles (CD8α, CD8β, FoxP3, IL-6, IFN-γ, perforin, RANTES) were analysed in a test cohort by immunohistochemistry and quantitative RT-PCR analysis on serial non-tumorous and tumorous tissues. Immune micro-environment was more inflammatory in HCV-HCC than HCV cirrhotic livers. The number of CD3(+) , CD4(+) , CD8(+) and CD20(+) liver-infiltrating lymphocytes was significantly higher, whereas the number of CD56(+) cells was significantly lower in HCV-HCC compared to HCV cirrhotic parenchyma. These differences were restricted to fibrous septa for CD4(+) and CD20(+) cells and to nodular parenchyma for CD8(+) cells. Gene expressions of CD8α, FoxP3 and RANTES were also significantly higher in HCV-HCC than in HCV cirrhosis. Interestingly, in a large cohort of 63 HCV-HCC patients. The number of CD8(+) cells ≥100/field was associated with significant higher tumour recurrence (P = 0.003) and lower overall survival (P = 0.05) at 5 years. High densities of liver-infiltrating lymphocytes in HCV-HCC cirrhotic parenchyma prevail inflammatory conditions and could contribute to tumorigenesis and tumour recurrence. These results could contribute towards better clinical evaluation of patients susceptible for HCC recurrence after curative surgery.

Human Cytotoxic T Lymphocyte-Mediated Acute Liver Failure and Rescue by Immunoglobulin in Human Hepatocyte Transplant TK-NOG Mice.

Hepatitis B virus (HBV)-specific cytotoxic T lymphocytes (CTLs) are critical in eliminating infection. We developed an animal model in which HBV-infected human hepatocytes are targeted by HBV-specific CTLs. After HBV inoculation in human hepatocyte-transplanted herpes simplex virus type-1 thymidine kinase-NOG mice, human peripheral blood mononuclear cells (PBMCs) were administered, and albumin, HBV DNA, alanine aminotransferase (ALT), and cytokine levels were analyzed. Histopathological and flow-cytometric analysis of infiltrating human immune cells were performed, and the efficacy of CTL-associated antigen-4 immunoglobulin (CTLA4Ig) against liver damage was evaluated. PBMC treatment resulted in massive hepatocyte damage with elevation of ALT, granzyme A, and gamma interferon and decrease in albumin and HBV DNA. The number of liver-infiltrating human lymphocytes and CD8-positive cells was significantly higher in HBV-infected mice. HBV-specific CTLs were detected by core and polymerase peptide-major histocompatibility complex-tetramer, and the population of regulatory T cells was significantly decreased in HBV-infected mice. Serum hepatitis B surface (HBs) antigen became negative, and HBs antibody appeared. CTLA4Ig treatment strongly inhibited infiltration of mononuclear cells. CTLA4Ig treatment will be used to treat patients who develop severe acute hepatitis B to prevent liver transplantation or lethality. This animal model is useful for virological and immunological analysis of HBV infection and to develop new therapies for severe acute hepatitis B. Without liver transplantation, some HBV-infected patients will die from severe liver damage due to acute overreaction of the immune system. No effective treatment exists, due in part to the lack of a suitable animal model. An animal model is necessary to investigate the mechanism of hepatitis and to develop therapeutic strategies to prevent acute liver failure in HBV infection. We developed an animal model in which HBV-infected human hepatocytes are targeted by human HBV-specific CTLs. In this model, HBV-infected human hepatocytes were transplanted into severely immunodeficient NOG mice in order to reconstruct elements of the human immune system. Using this model, we found that CTL-associated antigen-4 immunoglobulin was able to suppress damage to HBV-infected hepatocytes, suggesting an approach to treatment. This animal model is useful for virological and immunological analysis of HBV infection and to develop new therapies for severe acute hepatitis B.

Abstract B10: LFA-1/ICAM-1 interaction switches on an orchestrated prometastatic microenvironmental shift during experimental liver metastasis of colon C26 cancer cells.

Abstract Intercellular adhesion molecule (ICAM)-1 is the main counter-receptor for the lymphocyte function associated antigen (LFA)-1. However, the role this interaction plays during liver metastasis of colorectal cancer cells and its underlying mechanisms remain unclear. Previously, we showed that mannose receptor (ManR) stimulation on liver sinusoidal endothelial cells (LSECs) resulted in a reduced cytotoxic potential of liver infiltrating lymphocytes (LIL) towards C26 murine colon carcinoma cells. Using ICAM-1 specific siRNAs in vivo, we aimed to study the role of hepatic ICAM-1 in the prometastatic shift of the liver microenvironment. Livers of control and silenced mice, 24 hours and 14 days after C26 cell inoculation, were compared for: percentage of detained C26 cells, number and volume of metastatic foci, levels of recruited immune, myofibroblastic and inflammatory cells and whole liver expression of pro-inflammatory genes. Detained cancer cells and metastatic foci number were reduced in silenced mice compared to controls, along with a decrease in myeloid suppressor cells and recruited macrophages and myofibroblasts. On the contrary, significant higher numbers of lymphocytic cells were observed in mice with reduced ICAM-1. Finally, silencing of ICAM1 resulted in altered total liver mRNA levels of proinflammatory factors in the tumor-bearing mice. Therefore, an orchestrated prometastatic program might be switched on in the liver microenvironment by LFA-1/ICAM-1 interaction which, in turn, could lead not only to a reduced antitumor citotoxicity but, also, to activation of the stromal compartment favoring tumor progression, and, opening liver's doors to tumor cells to aggressively metastasize. Based on these results, LFA-1/ICAM-1 interaction arises as a potential therapeutic target in cancer treatment. Citation Format: Aitor Benedicto, Joana Marquez, Elvira Olaso, Beatriz Arteta. LFA-1/ICAM-1 interaction switches on an orchestrated prometastatic microenvironmental shift during experimental liver metastasis of colon C26 cancer cells.. [abstract]. In: Abstracts: AACR Special Conference on Cellular Heterogeneity in the Tumor Microenvironment; 2014 Feb 26-Mar 1; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2015;75(1 Suppl):Abstract nr B10. doi:10.1158/1538-7445.CHTME14-B10

Hepatic compartmentalization of exhausted and regulatory cells in HIV/HCV-coinfected patients.

Accelerated intrahepatic hepatitis C virus (HCV) pathogenesis is likely the result of dysregulation within both the innate and adaptive immune compartments, but the exact contribution of peripheral blood and liver lymphocyte subsets remains unclear. Prolonged activation and expansion of immunoregulatory cells have been thought to play a role. We determined immune cell subset frequency in contemporaneous liver and peripheral blood samples from chronic HCV-infected and HIV/HCV-coinfected individuals. Peripheral blood mononuclear cells (PBMC) and biopsy-derived liver-infiltrating lymphocytes from 26 HIV/HCV-coinfected, 10 chronic HCV-infected and 10 HIV-infected individuals were assessed for various subsets of T and B lymphocytes, dendritic cell, natural killer (NK) cell and NK T-cell frequency by flow cytometry. CD8(+) T cells expressing the exhaustion marker PD-1 were increased in HCV-infected individuals compared with uninfected individuals (P = 0.02), and HIV coinfection enhanced this effect (P = 0.005). In the liver, regulatory CD4(+) CD25(+) Foxp3(+) T cells, as well as CD4(+) CD25(+) PD1(+) T cells, were more frequent in HIV/HCV-coinfected than in HCV-monoinfected samples (P < 0.001). HCV was associated with increased regulatory T cells, PD-1(+) T cells and decreased memory B cells, regardless of HIV infection (P ≤ 0.005 for all). Low CD8(+) expression was observed only in PD-1(+) CD8(+) T cells from HCV-infected individuals and healthy controls (P = 0.002) and was associated with enhanced expansion of exhausted CD8(+) T cells when exposed in vitro to PHA or CMV peptides. In conclusion, in HIV/HCV coinfection, ongoing HCV replication is associated with increased regulatory and exhausted T cells in the periphery and liver that may impact control of HCV. Simultaneous characterization of liver and peripheral blood highlights the disproportionate intrahepatic compartmentalization of immunoregulatory T cells, which may contribute to establishment of chronicity and hepatic fibrogenesis in HIV coinfection.

Changes of regulatory T cells related to CCl₄-induced liver fibrosis in mice

To investigate liver fibrosis-related changes of CD4⁺CD25⁺Foxp3+ regulatory T cells (Tregs) in peripheral blood and in liver-infiltrating lymphocytes (LILs) using a mouse model. C57BL/6 mice were randomly divided into a model group and a control group. The model group received intraperitoneal injection of carbon tetrachloride (CC1₄) to induce liver fibrosis, and the control group received an equal volume of physiological saline. Serum was collected for detection of alanine aminotransferase level. Histopathological changes in liver were assessed by microscopic observation of tissues stained by hematoxylineosin and Masson. Frequencies of peripheral and intrahepatic Tregs, NK1.1⁺ cells, CD4⁺ and CD8⁺ cells were analyzed by flow cytometry. Intrahepatic Foxp3+ cells were detected by immunofluorescence. Liver expression of IL-6, IL-10, TGFb and Foxp3 was measured by RT-PCR detection of mRNA .Inter-group differences were evaluated by t-test. The model group showed a significantly higher frequency of intrahepatic CD25⁺Foxp3⁺/CD4⁺ Tregs (10.63% ± 1.50% vs. control group:1.80% ± 0.66%; P less than 0.01) but only slightly higher frequency of peripheral Tregs (6.00% ± 0.62% vs. 5.33% ± 2.86%). The model group also showed significantly higher levels of intrahepatic Foxp3+ cells and of Foxp3 mRNA (both P less than 0.05), but significantly lower frequencies of NK1.1 cells in LILs (9.53% ± 2.25% vs. 19.80% ± 5.97%; P less than 0.05) and in peripheral blood (0.38% ± 0.13% vs. 1.06% ± 0.63%; P less than 0.05). The CD4⁺ cell frequency and the CD4⁺/CD8⁺ ratio were lower in LILs and peripheral blood of the model group, but none differed significantly from the control group. The intrahepatic expression of TGFb mRNA was significantly higher in the model group (P less than 0.01). In liver fibrosis, intrahepatic CD4⁺CD25⁺Foxp3+ Tregs are increased while NK1.1+ cells are decreased. Tregs may suppress NK1.1+ cells through a mechanism involving TGFb.

HCV adaptations to altered CD8+ T-cell immunity during pregnancy.

Robust, polyfunctional CD8+ T-cell responses targeted to multiple viral epitopes have been associated with successful resolution of acute HCV infection. In most individuals, however, immunity fails and chronic viremia ensues as CD8+ T cells become functionally exhausted or select viral variants with escape mutations in class I epitopes. During pregnancy, dysfunctional HCV-specific CD8+ T-cell responses may be further impaired by maternal–fetal tolerance processes such as the expansion of regulatory T-cell populations or hormonal changes. Recent evidence suggests that HCV adapts to relaxed maternal CD8+ T-cell pressure in pregnancy by shedding unfit escape mutations in certain class I epitopes, resulting in the selection of viruses with more efficient replication. Emergence of HCV variants with enhanced replicative capacity during pregnancy has ramifications for mother-to-child transmission, the primary route of HCV infection in children, and could potentially be relevant to other important, vertically transmissible, persistent viruses such as HBV and HIV. HCV has infected over 185 million people worldwide and persists in approximately 75% of these individuals, predisposing to liver inflammation, cirrhosis and hepatocellular carcinoma [1]. More than 1 million children are born to HCV-infected mothers each year, and 3–5% acquire the infection in utero or at delivery, making vertical transmission the leading route of pediatric HCV infection in the developed world [2]. Here, we review the critical role of CD8+ T-cell immunity in HCV infection, its modulation in pregnancy and recent evidence that HCV takes advantage of the unique immunologic niche of pregnancy to improve replicative fitness and potentially promote vertical transmission.

Intrahepatic interleukin‐17+T cells and FoxP3+ regulatory T cells cooperate to promote development and affect the prognosis of hepatocellular carcinoma

Recent studies have shown that imbalance between tumor-infiltrating interleukin (IL)-17(+) T cells and regulatory T cells (Tregs) is an important regulator of progression in various cancers, but little is known regarding this imbalance in hepatocellular carcinoma (HCC). This study explored the role of imbalance between IL-17(+) T cells and Tregs in the immunopathogenesis of HCC in patients with chronic hepatitis B (CHB) infection. Fifty-six of patient-matched tumors and peritumoral surgical specimens from 56 patient with HCC and 136 liver biopsies specimens from 46 patients with CHB, 37 with atypical hyperplasia (AH), and 53 with HCC were enrolled. The expressions of IL-17, FoxP3, CD4, and CD8 in liver tissue were measured by immunochemistry for the evaluation of liver-infiltrating lymphocytes. The density of liver infiltrated FoxP3(+) Tregs was increased in a stepwise manner from CHB to AH then HCC, while there was a decreasing trend for the density of IL-17(+) T cells and CD8(+) T cells. In surgical specimens of less differentiated HCC, the quantity of tumor-infiltrating FoxP3(+) Tregs was significantly lower and IL-17(+) T cells and CD8(+) T cells were significantly higher. Additionally, peritumoral IL-17(+) T cells were increased in poorly differentiated HCC. High intratumoral FoxP3(+) Tregs with high intratumoral IL-17(+) T cells showed a significantly lower overall survival (OS) and disease-free survival (DFS) compared with other groups (OS, P = 0.033; DFS, P = 0.004). High intratumoral FoxP3(+) Tregs with high peritumoral IL-17(+) T cells showed a significantly lower survival rate compared with other groups (OS, P < 0.001 and DFS, P < 0.001). Our findings suggest that intrahepatic IL-17(+) T cells and FoxP3(+) Tregs may cooperate to promote the progression of HCC.

Low‐dose anti‐CD3 antibody induces remission of active autoimmune hepatitis in xenoimmunized mice

Some patients with autoimmune hepatitis (AIH), despite appropriate treatment, progress towards cirrhosis and liver failure, requiring transplantation. New biological agents targeting immune cell subtypes have been developed, with better specificity and longer-lasting effects than conventional wide-spectrum immunosuppressive drugs. The goal of this study was to evaluate the effectiveness of low dose of αCD3 targeting therapy in a model of type 2 AIH. This experimental model is based on xenoimmunization of C57BL/6 mice with DNA coding for human liver autoantigens. Mice with AIH were treated with five daily injections of low dose of αCD3 monoclonal antibody, before disease onset (5.5 months post-xenoimmunization) or during AIH (7 months post-xenoimmunization). Along with serum aminotransferases, autoantibody levels and end-point liver histology, spleen and liver-infiltrating lymphocytes were phenotyped by flow cytometry and immune response measured by lymphoproliferative assays. Before onset of AIH, treatment prevented the development of liver inflammation and tissue injury. During active AIH, low dose of αCD3 antibody therapy resulted in a resorption of liver inflammatory infiltrates, normalization of serum aminotransferas levels, reduced autoantibody titres, increased regulatory T cells and lowered proliferation of autoreactive liver lymphocytes. We report that low dose αCD3 antibody administration is an effective treatment for AIH in an experimental model of type 2 AIH. These data suggest that αCD3 antibody therapy could be tested in clinical trials as a rescue therapy for patients with uncontrolled AIH.

Increased IL-22-producing cells contribute to liver fibrosis through promoting Th17 migration in chronic HBV patients (P3363)

Abstract Interleukin-22 is markedly upregulated in chronic HBV (CHB) infection; however, its functional role in patients with liver fibrosis (LC) remains obscure. We screened mRNA expression in HBV-infected liver tissue vs normal control and found IL-22-associated pathway was significantly changed. We further found that intrahepatic IL-22+ cell counts were significantly increased in HBV-infected patients, especially in LC patients. Further analysis indicated that IL-22 could be produced by various liver-infiltrating lymphocytes, but preferentially produced by IL-22+IL-17a+IFN-γ- Th17 cells in LC patients, which was also positively associated with liver fibrosis severity in CHB patients. We also found human HSCs express high levels of IL-22R. IL-22 exposure increased pSTAT3 expression and fibrotic protein production, and also prevented soluble TRAIL-mediated HSC apoptosis in vitro. Blockade of IL-22 in a HBV-transgenic mice model with T cell-mediated liver fibrosis significantly restricted liver fibrosis progression, and Th17 migration and CCL20 expression. IL-22-treated HSCs secreted CCL20, and blockade of CCL20 preferentially reduced the migration of Th17 cells in vitro. These data suggest that the increased IL-22-producing cells contribute to liver fibrosis through promoting Th17 migration in chronic HBV patients, and may facilitate the rational development of immunotherapeutic strategies targeting IL-22 for the amelioration of liver fibrosis in chronically HBV-infected subjects.

T cell receptor variable β gene repertoire in liver and peripheral blood lymphocytes of chronically hepatitis C virus-infected patients with and without mixed cryoglobulinaemia

To characterize the repertoire of T lymphocytes in chronically hepatitis C virus (HCV)-infected patients with and without mixed cryoglobulinaemia (MC). T cell receptor (TCR) variable (V) β clonalities in portal tracts isolated from liver biopsy sections with a laser capture microdissection technique in 30 HCV-positive MC patients were studied by size spectratyping. Complementarity-determining region 3 (CDR3) profiles of liver-infiltrating lymphocytes (LIL) were also compared with those circulating in the blood. The representative results of TCR Vβ by CDR3 were also obtained from liver tissues and peripheral blood lymphocytes (PBL) of 21 chronically HCV-infected patients without MC. LIL were highly restricted, with evidence of TCR Vβ clonotypic expansions in 23 of 30 (77%) and in 15 of 21 (71%) MC and non-MC patients, respectively. The blood compartment contained TCR Vβ expanded clones in 19 (63%) MC and 12 (57%) non-MC patients. The occurrence of LIL clonalities was detected irrespective of the degree of liver damage or circulating viral load, whereas it correlated positively with higher levels of intrahepatic HCV RNA. These results support the notion that TCR Vβ repertoire is clonally expanded in HCV-related MC with features comparable to those found in chronically HCV-infected patients without MC.

Plasmacytoid dendritic cells induce efficient stimulation of antiviral immunity in the context of chronic hepatitis B virus infection

The immune control of hepatitis B virus (HBV) infection is essential for viral clearance. Therefore, restoring functional anti-HBV immunity is a promising immunotherapeutic approach to treatment of chronic infection. Plasmacytoid dendritic cells (pDCs) play a crucial role in triggering antiviral immunity through their ability to capture and process viral antigens and subsequently induce adaptive immune responses. We investigated the potential of pDCs to trigger antiviral cellular immunity against HBV. We used a human leukocyte antigen A (HLA-A)*0201(+) pDC line loaded with HLA-A*0201-restricted peptides derived from hepatitis B core/hepatitis B surface (HBc/HBs) antigens to amplify specific CD8 T cells ex vivo from chronic HBV patients and established a Hepato-HuPBL mouse model to address the therapeutic potential of the strategy in vivo. Stimulation of PBMCs or liver-infiltrating lymphocytes from HLA-A*0201(+) chronic HBV patients by HBc peptide-loaded pDCs elicited up to 23.1% and 76.1% HBV-specific CD8 T cells in 45.8% of cases. The specific T cells from the "responder" group secreted interferon-γ, expressed CD107 upon restimulation, and efficiently lysed HBV antigen-expressing hepatocytes. Circulating hepatitis B e antigen (HBeAg) was found to distinguish the group of patients not responding to the pDC stimulation. The therapeutic efficacy of the pDC vaccine was evaluated in immunodeficient NOD-SCID β(2) m(-/-) mice reconstituted with HBV patients' PBMCs and xenotransplanted with human HBV-transfected hepatocytes. Vaccination of Hepato-HuPBL mice with the HBc/HBs peptide-loaded pDCs elicited HBV-specific T cells able to specifically lyse the transfected hepatocytes and reduce the systemic viral load. pDCs loaded with HBV-derived peptides can elicit functional virus-specific T cells. HBeAg appears to be critical in determining the outcome of immunotherapies in chronic HBV patients. A pDC-based immunotherapeutic approach could be of interest in attempts to restore functional antiviral immunity, which is critical for the control of the virus in chronic HBV patients.

Are non‐traditional CD4+CD69+CD25– regulatory T cells involved in disease progression of human hepatocellular carcinoma?

See article in J. Gastroenterol. Hepatol. 2011; 26: 1519–1526.

P89 Osteopontin promotes lymphocyte recruitment in steatohepatitis

IntroductionSteatohepatitis is the critical step in the progression to fibrosis, and is characterised by increased inflammatory cell recruitment from the circulation. The cytokine Osteopontin (OPN) is intricately involved in cell-recruitment...

The balance between intrahepatic IL-17+ T cells and Foxp3+ regulatory T cells plays an important role in HBV-related end-stage liver disease

BackgroudIL-17+ T helper cells and Foxp3+ regulatory T cells are CD4+ T helper cells with reciprocally regulated differentiation and function. Their frequency and function vary in patients with chronic hepatitis B. In this study, we investigated the balance between IL-17+ T cells and Foxp3+ regulatory T cells and illustrated their function in the aggravation of chronic hepatitis B (CHB).ResultsTwenty-six patients with chronic HBV -related liver failure (CLF), thirty-one patients with acute on chronic HBV-related liver failure (ACLF) and twelve normal controls were enrolled in our study. The expressions of IL-17, Foxp3, CD4, CD8 and perforin in liver tissue were measured by immunochemistry for the evaluation of liver-infiltrating lymphocytes. The frequency of liver IL-17+ T cells on liver inflammatory cells and their proportion in the total CD4+ T cell population increased markedly in the ACLF group, while the frquency of Foxp3+ T cells and their proportion in the total CD4+ T cell population did not show a significant difference in the two HBV infection groups. In addition, the ACLF group showed a dramatically higher IL-17+ /Foxp3+ ratio than the CLF group. CD4+ T cells increased significantly in the liver of patients with ACLF, compared with those in the liver of patients with CLF.ConclusionsOur findings suggest that intrahepatic IL-17+ T cells play an important role in the development of chronic HBV and that the imbalance between IL-17+ and Foxp3+ T cells in the liver may lead to progression of the disease but the mechanism should be further explored.

Immunostaining of PD-1/PD-Ls in liver tissues of patients with hepatitis and hepatocellular carcinoma

To investigate the expression of programmed death (PD)-1, PD ligand 1 (PD-L1) and PD-L2 in liver tissues in the context of chronic hepatitis and hepatocellular carcinoma (HCC). Liver biopsies and HCC specimens from patients were collected and histologically examined. The expression of PD-1, PD-L1, and PD-L2 in biopsy specimens of chronic hepatitis and HCC specimens was evaluated by immunohistochemical staining. The association between the expression level of PD-1, PD-L1, and PD-L2 and clinical and pathological variables was analyzed statistically. Expression of PD-1 was found in liver-infiltrating lymphocytes. In contrast, PD-L1 and PD-L2 were expressed in non-parenchyma liver cells and tumor cells. The expression of PD-L1 was significantly correlated with hepatitis B virus infection (1.42 ± 1.165 vs 0.50 ± 0.756, P = 0.047) and with the stage of HCC (7.50 ± 2.121 vs 1.75 ± 1.500 vs 3.00 ± 0.001, P = 0.018). PD-1 and PD-Ls were significantly up-regulated in HCC specimens (1.40 ± 1.536 vs 5.71 ± 4.051, P = 0.000; 1.05 ± 1.099 vs 4.29 ± 3.885, P = 0.004; 1.80 ± 1.473 vs 3.81 ± 3.400, P = 0.020). PD-L1 may contribute to negative regulation of the immune response in chronic hepatitis B. PD-1 and PD-Ls may play a role in immune evasion of tumors.