Primary biliary cirrhosis (PBC) is considered a model autoimmune disease, with the most highly directed and specific autoantibody in both murine and human autoimmunity, the anti-mitochondrial autoantibody (AMA). However, therapeutic advances in this disease have lagged behind. Herein we have taken advantage of our unique model of murine PBC in which mice immunized with 2-octynoic acid coupled to BSA (2OA-BSA), a compound identified by quantitative structure activity relationships (QSAR) of human AMA binding, develop an intense inflammatory cholangitis with striking similarities to humans with PBC. In particular, we have constructed several unique gene-deleted mice, including mice deleted of IL-12p40, IL-12p35, IFN-γ, IL-23p19, IL-17A, IL-17F and IL-22, immunized these animals with 2OA-BSA and followed the natural history of immunopathology to identify key pathways that might provide clues for successful therapy. Our data indicate that whereas both IL-12/Th1 and IL-23/Th17 are involved in cholangitis, it is the IL-12/Th1 signaling pathway that elicits pathology. In fact, deletion of IFN-γ prevents disease and suppresses autoantibodies. Importantly, deletion of the Th17 cytokines IL-17A and IL-22, but not IL-17F, reduces biliary damage; IL-17A-knockout mice have reduced levels of anti-mitochondrial antibody. We further demonstrate that the production of IFN-γ is significantly decreased in the liver of IL-23p19−/−, IL-17A−/− and IL-22−/− mice compared with controls. However, the ability of T cells to produce IFN-γ was not affected in Th17 cytokine-deficient mice. Our data indicate that a deficient Th17 pathway suppresses the accumulation of IFN-γ producing cells in liver during the early phase of cholangitis. In conclusion, whereas IFN-γ has a pivotal role in the early events involved in the pathogenesis of autoimmune cholangitis induced by 2OA-BSA, the IL-23/Th17 pathway potentiates the effects of IL-12/IFN-γ-mediated immunopathology.
The human antibody repertoire is one of the most important defenses against infectious disease, and the development of vaccines has enabled the conferral of targeted protection to specific pathogens. However, there are many challenges to measuring and analyzing the immunoglobulin sequence repertoire, such as the fact that each B cell contains a distinct antibody sequence encoded in its genome, that the antibody repertoire is not constant but changes over time, and the high similarity between antibody sequences. We have addressed this challenge by using high-throughput long read sequencing to perform immunogenomic characterization of expressed human antibody repertoires in the context of influenza vaccination. Informatic analysis of 5 million antibody heavy chain sequences from healthy individuals allowed us to perform global characterizations of isotype distributions, determine the lineage structure of the repertoire and measure age and antigen related mutational activity. Our analysis of the clonal structure and mutational distribution of individuals’ repertoires shows that elderly subjects have a decreased number of lineages but an increased pre-vaccination mutation load in their repertoire and that some of these subjects have an oligoclonal character to their repertoire in which the diversity of the lineages is greatly reduced relative to younger subjects. We have thus shown that global analysis of the immune system’s clonal structure provides direct insight into the effects of vaccination and provides a detailed molecular portrait of age-related effects.
There has been increased interest in the role of B cells in the pathogenesis of primary biliary cirrhosis. Although the vast majority of patients with primary biliary cirrhosis have antimitochondrial antibodies, there is no correlation of antimitochondrial antibody titer and/or presence with disease severity. Further, in murine models of primary biliary cirrhosis, it has been suggested that depletion of B cells may exacerbate biliary pathology. To address this issue, we have focused on detailed phenotypic characterization of mononuclear cell infiltrates surrounding the intrahepatic bile ducts of patients with PBC, PSC, AIH, CH-C and GVHD, including CD3, CD4, CD8, CD20, CD38 and immunoglobulin classes, as well as double immunohistochemical staining for CD38 and IgM. Interestingly, CD20 B lymphocytes, which are a precursor of plasma cells, were found in scattered locations or occasionally forming follicle-like aggregations but were not noted at the proximal location of chronic nonsuppurative destructive cholangitis. In contrast, there was a unique and distinct coronal arrangement of CD38 cells around the intrahepatic ducts in primary biliary cirrhosis but not controls; the majority of such cells were considered plasma cells based on their expression of intracellular immunoglobulins, including IgM and IgG, but not IgA. Patients with primary biliary cirrhosis who manifest this unique coronal arrangement were those with significantly higher titers of antimitochondrial antibodies. These data collectively suggest a role of plasma cells in the specific destruction of intrahepatic bile ducts in primary biliary cirrhosis and highlight the increasing interest in plasma cells and autoimmunity.
antimitochondrial antibodies; primary biliary cirrhosis; plasma cells; coronal arrangement
A major enigma of primary biliary cirrhosis (PBC) is the selective targeting of biliary cells. Our laboratory has reported that following apoptosis human intrahepatic biliary epithelial cells (HiBEC) translocate the E2 subunit of the pyruvate dehydrogenase complex immunologically intact into apoptotic bodies, forming an apotope. However, the cell type and specificity of this reaction has not been fully defined. To address this issue we have investigated whether PDC-E2, BCOADC-E2, OGDC-E2, four additional inner mitochondrial enzymes and four nuclear antigens remain immunologically intact with respect to post-apoptotic translocation in HiBEC and 3 additional control epithelial cells. We report that all three 2-oxo acid dehydrogenase enzymes share the ability to remain intact within the apotope of HiBEC. Interestingly the E2 subunit of the branched chain 2-oxo acid dehydrogenase complex also remained intact in the other cell types tested. We extended the data using 95 AMA+ and 19 AMA- PBC and 76 control sera for reactivity against the 7 mitochondrial proteins studied herein and also the ability of AMA- sera to react with HIBEC apotopes. Sera from 3/95 AMA+ sera, but none of the controls, reacted with 2, 4-dienoyl CoA reductase 1 (DECR1), an enzyme also present intact only in the HiBEC apotope; DECR1 has not been previously associated with any autoimmune disease. Finally the specificity of HIBEC apotope reactivity was confined to AMA+ sera. In conclusion, we submit that the biliary specificity of PBC is secondary to the unique processes of biliary apoptosis.
Apoptosis; human intrahepatic biliary epithelial cells; E2 subunit of 2-oxoacid dehydrogenase complex; nuclear antigens; autoimmunity
The rejection of unfair offers can be affected by both negative emotions (e.g. anger and moral disgust) and deliberate cognitive processing of behavioral consequences (e.g. concerns of maintaining social fairness and protecting personal reputation). However, whether negative emotions are sufficient to motivate this behavior is still controversial. With modified ultimatum games, a recent study (Yamagishi T, et al. (2009) Proc Natl Acad Sci USA 106∶11520–11523) found that people reject unfair offers even when this behavior increases inequity, and even when they could not communicate to the proposers. Yamagishi suggested that rejection of unfair offers could occurr without people’s concerning of maintaining social fairness, and could be driven by negative emotions. However, as anonymity was not sufficiently guaranteed in Yamagishi’s study, the rejection rates in their experiments may have been influenced by people’s concerns of protecting personal reputation (reputational concerns) in addition to negative emotions; thus, it was unclear whether the rejection was driven by negative emotions, or by reputational concerns, or both. In the present study, with specific methods to ensure anonymity, the effect of reputational concerns was successfully ruled out. We found that in a private situation in which rejection could not be driven by reputational concerns, the rejection rates of unfair offers were significantly larger than zero, and in public situations in which rejection rates could be influenced by both negative emotions and reputational concerns, rejection rates were significantly higher than that in the private situation. These results, together with Yamagishi’s findings, provided more complete evidence suggesting (a) that the rejection of unfair offers can be driven by negative emotions and (b) that deliberate cognitive processing of the consequences of the behavior can increase the rejection rate, which may benefit social cooperation.
Conventional measurement of antibody responses to vaccines largely relies on serum antibodies, which are primarily produced by bone marrow plasma cells and may not represent the entire vaccine-induced B cell repertoire, including important functional components such as those targeted to mucosal sites. After immunization or infection, activated B cells differentiate into plasmablasts in local lymphoid organs, then traffic through circulation to the target sites where they further develop into plasma cells. On day 7 after influenza vaccination, a burst of plasmablasts, highly enriched for vaccine-specific antibody secreting cells, appears in the peripheral blood. This provides a unique window to the overall B cell response to the vaccine, without interference of pre-existing cross-reactive serum antibody. In this study we isolated B cells from volunteers on day 7 after immunization with the inactivated influenza vaccine and cultured them ex vivo to collect plasmablast-derived polyclonal antibodies (PPAb). The PPAb contained secreted IgG and IgA, which was approximately 0.2 ng per antibody secreting cell. Influenza-specific IgG and IgA binding activity was detected in PPAb at dilutions up to 105 by ELISA. The ratio of the titers of influenza-specific IgA to IgG by ELISA was 4-fold higher in PPAb than in day 28 post-vaccination sera, suggesting that vaccine-induced IgA is enriched in PPAb compared to sera. Functional activity was also detected in PPAb as determined by microneutralization and hemagglutination inhibition assays. In addition to bulk B cell cultures, we also cultured plasmablast subsets sorted by cell surface markers to generate PPAb. These results suggest that PPAb better reflects the mucosal IgA response than serum samples. Since PPAb are exclusively produced by recently activated B cells, it allows assessing vaccine-induced antibody response without interference from pre-existing cross-reactive serum antibodies and permits an assessment of antibody avidity based on antigen specific binding and antibody quantity. Therefore this assay is particularly useful for studying vaccine/infection-induced antibodies against antigens that might have previously circulated, such as antibody responses to rotavirus, dengue or influenza viruses in which cross-reactive antibodies against different virus serotypes/subtypes play a critical role in immunity and/or pathogenesis.
Influenza virus; vaccines; antibodies; plasmablasts
During seasonal influenza epidemics, disease burden is shouldered predominantly by the very young and the elderly. Elderly individuals are particularly affected, in part because vaccine efficacy wanes with age. This has been linked to a reduced ability to induce a robust serum antibody response. Here, we show that this is due to reduced quantities of vaccine-specific antibodies, rather than a lack of antibody avidity or affinity. We measured levels of vaccine-specific plasmablasts by ELISPOT 1 week after immunization of young and elderly adults with inactivated seasonal influenza vaccine. Plasmablast-derived polyclonal antibodies (PPAbs) were generated from bulk-cultured B cells, while recombinant monoclonal antibodies (re-mAbs) were produced from single plasmablasts. The frequency of vaccine-specific plasmablasts and the concentration of PPAbs were lower in the elderly than in young adults, whereas the yields of secreted IgG per plasmablast were not different. Differences were not detected in the overall vaccine-specific avidity or affinity of PPAbs and re-mAbs between the 2 age groups. In contrast, reactivity of the antibodies induced by the inactivated seasonal influenza vaccine toward the 2009 pandemic H1N1 virus, which was not present in the vaccine, was higher in the elderly than in the young. These results indicate that the inferior antibody response to influenza vaccination in the elderly is primarily due to reduced quantities of vaccine-specific antibodies. They also suggest that exposure history affects the cross-reactivity of vaccination-induced antibodies.
Interferon (IFN) plays a central role in the innate and adaptive antiviral immune responses. While IFN-α is currently approved for treating chronic hepatitis B and hepatitis C, in limited studies, IFN-γ has not been shown to be effective for chronic hepatitis B or C. To identify the potential mechanism underlying the differential antiviral effects of IFN-α and IFN-γ, we used cDNA microarray to profile the global transcriptional response to IFN-α and IFN-γ in primary human hepatocytes, the target cell population of hepatitis viruses. Our results reveal distinct patterns of gene expression induced by these 2 cytokines. Overall, IFN-α induces more genes than IFN-γ at the transcriptional level. Distinct sets of genes were induced by IFN-α and IFN-γ with limited overlaps. IFN-α induces gene transcription at an early time point (6 h) but not at a later time point (18 h), while the effects of IFN-γ are more prominent at 18 h than at 6 h, suggesting a delayed transcriptional response to IFN-γ in the hepatocytes. These findings indicate differential actions of IFN-α and IFN-γ in the context of therapeutic intervention for chronic viral infections in the liver.
There have been several descriptions of mouse models that manifest select immunological and clinical features of autoimmune cholangitis with similarities to primary biliary cirrhosis in humans. Some of these models require immunization with complete Freund's adjuvant, whereas others suggest that a decreased frequency of T regulatory cells (Tregs) facilitate spontaneous disease. We hypothesized that anti-mitochondrial antibodies (AMA) and development of autoimmune cholangitis would be found in mice genetically deficient in components essential for the development and homeostasis of Foxp3+ Tregs. Therefore, we examined Scurfy (Sf) mice, animals that have a mutation in the gene encoding the Foxp3 transcription factor which results in a complete abolition of Foxp3+ Tregs. Indeed at 3-4 weeks of age, 100% of animals manifest high titer serum AMA of all isotypes. Further, mice have moderate to severe lymphocytic infiltrates surrounding portal areas with evidence of biliary duct damage, and dramatic elevation of cytokines in serum and mRNAs encoding cytokines in liver tissue, including TNF-α, IFN-γ, IL-6, IL-12 and IL-23. In conclusion, the lack of functional Foxp3 is a major predisposing feature for loss of tolerance that leads to autoimmune cholangitis. These findings reflect on the importance of regulatory T cells in other murine models as well as in patients with PBC.
Sf mouse; Primary Biliary Cirrhosis; Tregs; Interleukin 12; CD8 T lymphocyte
Rotavirus (RV) infection of the intestine is the major cause of severe dehydrating diarrhea in infants around the world. Although protective immunity against RV, especially acquired B and T cell responses, have been extensively studied, our understanding of RV immunity remains incomplete. In addition, the interaction between various protective immune mechanisms in the gut and specific enteric immune suppressor systems that normally exert a regulatory function on mucosal immunity has not been extensively investigated. Among the candidate suppressor systems, we hypothesized that CD4+ CD25+ Foxp3+ regulatory T (Treg) cells may play a role in modulating RV immunity since such cells are naturally present in large numbers in the intestine and function nonspecifically. Here we demonstrate that neonatal murine RV (EC) infection induces an expansion of the Treg cell population and the magnitude of the T cell mediated immune response is modulated by Treg cells. Accordingly, when natural Treg cells in neonatal mice were depleted before virus infection, both CD4+ and CD8+ T cell responses to RV, such as proliferation and IFN-γ secretion, were enhanced in mesenteric lymph nodes (MLNs) and the spleen. Interestingly, increased proliferation of CD19+ B cells from Treg cell depleted animals was also observed. Finally, we analyzed the in vivo effect of the Treg cell depletion on diarrheal disease, virus shedding and IgA RV-specific response. Treg cell depletion did not affect these functions. Our studies of immune modulatory Treg cells in the RV infection model may promote a better understanding of the basis for RV immunity as well as providing valuable clues for the development of more immunogenic RV vaccines.
Rotavirus; Regulatory T cell; vaccines
CD8 T-cell response provides an important defense against rotavirus, which infects a variety of systemic locations in addition to the gut. Here we investigated the distribution, phenotype, and function of rotavirus-specific CD8 T cells in multiple organs after rotavirus infection initiated via the intranasal, oral, or intramuscular route. The highest level of virus-specific CD8 T cells was observed in the Peyer's patches of orally infected mice and in the lungs of intranasally infected animals. Very low levels of virus-specific CD8 T cells were detected in peripheral blood or spleen irrespective of the route of infection. Rotavirus-specific CD8 T cells from Peyer's patches of orally infected mice expressed high levels of CCR9, while CXCR6 and LFA-1 expression was associated with virus-specific CD8 T cells in lungs of intranasally infected mice. Oral infection induced the highest proportion of gamma interferon− CD107a/b+ CD8 T cells in Peyer's patches. When equal numbers of rotavirus-specific CD8 T cells were transferred into Rag-1 knockout mice chronically infected with rotavirus, the donor cells derived from Peyer's patches of orally infected mice were more efficient than those derived from lungs of intranasally infected animals in clearing intestinal infection. These results suggest that different routes of infection induce virus-specific CD8 T cells with distinct homing phenotypes and effector functions as well as variable abilities to clear infection.
Currently two vaccines, trivalent inactivated influenza vaccine (TIV) and live attenuated influenza vaccine (LAIV), are licensed in the USA. Despite previous studies on immune responses induced by these two vaccines, a comparative study of the influence of prior influenza vaccination on serum antibody and B-cell responses to new LAIV or TIV vaccination has not been reported. During the 2005/6 influenza season, we quantified the serum antibody and B-cell responses to LAIV or TIV in adults with differing influenza vaccination histories in the prior year: LAIV, TIV, or neither. Blood samples were collected on days 0, 7–9 and 21–35 after immunization and used for serum HAI assay and B-cell assays. Total and influenza-specific circulating IgG and IgA antibody secreting cells (ASC) in PBMC were detected by direct ELISPOT assay. Memory B cells were also tested by ELISPOT after polyclonal stimulation of PBMC in vitro. Serum antibody, effector, and memory B-cell responses were greater in TIV recipients than LAIV recipients. Prior year TIV recipients had significantly higher baseline HAI titers, but lower HAI response after vaccination with either TIV or LAIV, and lower IgA ASC response after vaccination with TIV than prior year LAIV or no vaccination recipients. Lower levels of baseline HAI titer were associated with a greater fold-increase of HAI titer and ASC number after vaccination, which also differed by type of vaccine. Our findings suggest that the type of vaccine received in the prior year affects the serum antibody and the B-cell responses to subsequent vaccination. In particular, prior year TIV vaccination is associated with sustained higher HAI titer one year later but lower antibody response to new LAIV or TIV vaccination, and a lower effector B-cell response to new TIV but not LAIV vaccination.
Factors affecting immune responses to influenza vaccines have not been studied systematically. We hypothesized that T-cell and antibody responses to the vaccines are functions of pre-existing host immunity against influenza antigens.
During the 2004 and 2005 influenza seasons, we have collected data on cellular and humoral immune reactivity to influenza virus in blood samples collected before and after immunization with inactivated or live attenuated influenza vaccines in healthy children and adults. We first used cross-validated lasso regression on the 2004 dataset to identify a group of candidate baseline correlates with T-cell and antibody responses to vaccines, defined as fold-increase in influenza-specific T-cells and serum HAI titer after vaccination. The following baseline parameters were examined: percentages of influenza-reactive IFN-γ+ cells in T and NK cell subsets, percentages of influenza-specific memory B-cells, HAI titer, age, and type of vaccine. The candidate baseline correlates were then tested with the independent 2005 dataset. Baseline percentage of influenza-specific IFN-γ+ CD4 T-cells was identified as a significant correlate of CD4 and CD8 T-cell responses, with lower baseline levels associated with larger T-cell responses. Baseline HAI titer and vaccine type were identified as significant correlates for HAI response, with lower baseline levels and the inactivated vaccine associated with larger HAI responses. Previously we reported that baseline levels of CD56dim NK reactivity against influenza virus inversely correlated with the immediate T-cell response to vaccination, and that NK reactivity induced by influenza virus depended on IL-2 produced by influenza-specific memory T-cells. Taken together these results suggest a novel mechanism for the homeostasis of virus-specific T-cells, which involves interaction between memory helper T-cells, CD56dim NK and DC.
These results demonstrate that assessment of baseline biomarkers may predict immunologic outcome of influenza vaccination and may reveal some of the mechanisms responsible for variable immune responses following vaccination and natural infection.
The patterns of cellular immune responses induced by live attenuated influenza vaccine (LAIV) versus those of the trivalent inactivated influenza vaccine (TIV) have not been studied extensively, especially in children. The goals of this study were to evaluate the effects of TIV and LAIV immunization on cellular immunity to live influenza A virus in children and adults and to explore factors associated with variations in responses to influenza vaccines among individuals. A gamma interferon (IFN-γ) flow cytometry assay was used to measure IFN-γ-producing (IFN-γ+) NK and T cells in peripheral blood mononuclear cell cultures stimulated with a live influenza A virus strain before and after LAIV or TIV immunization of children and adults. The mean percentages of influenza A virus-specific IFN-γ+ CD4 and CD8 T cells increased significantly after LAIV, but not TIV, immunization in children aged 5 to 9 years. No increases in the mean levels of influenza A virus-reactive IFN-γ+ T cells and NK cells were observed in adults given LAIV or TIV. TIV induced a significant increase in influenza A virus-reactive T cells in 6-month- to 4-year-old children; LAIV was not evaluated in this age group. The postvaccination changes (n-fold) in the percentages of influenza A virus-reactive IFN-γ+ T and NK cells in adults were highly variable and correlated inversely with the prevaccination percentages, in particular with that of the CD56dim NK cell subset. In conclusion, our findings identify age, type of vaccine, and prevaccination levels of immune reactivity to influenza A virus as factors significantly associated with the magnitude of cellular immune responses to influenza vaccines.
The role of human NK cells in viral infections is poorly understood. We used a cytokine flow-cytometry assay to simultaneously investigate the IFN-γ response of NK and T lymphocytes to influenza A virus (fluA). When PBMCs from fluA-immune adult donors were incubated with fluA, IFN-γ was produced by both CD56dim and CD56bright subsets of NK cells, as well as by fluA-specific T cells. Purified NK cells did not produce IFN-γ in response to fluA, while depletion of T lymphocytes reduced to background levels the fluA-induced IFN-γ production by NK cells, which indicates that T cells are required for the IFN-γ response of NK cells. The fluA-induced IFN-γ production of NK cells was suppressed by anti–IL-2 Ab, while recombinant IL-2 replaced the helper function of T cells for IFN-γ production by NK cells. This indicates that IL-2 produced by fluA-specific T cells is involved in the T cell–dependent IFN-γ response of NK cells to fluA. Taken together, these results suggest that at an early stage of recurrent viral infection, NK-mediated innate immunity to the virus is enhanced by preexisting virus-specific T cells.
Relatively little is known for the differentiation and maturation process of human B cells to plasma cells. This is particularly important in reconstitution work involving transfer of autoantibodies. To address this issue, we transplanted human peripheral blood mononuclear cells (PBMC) directly into the spleen of irradiated NOD/SCID mice depleted of natural killer cell activity. Within 6 weeks, naïve B cells differentiated into memory B cells and, importantly, the numbers of human CD138+ plasma cells in spleen increased by 100 fold after transplantation. Plasma cell numbers correlated with the detection of human IgM and IgG in serum, indicating that human B cells had differentiated into mature plasma cells in the murine spleen. In addition to CD19+ plasma cells, a distinct CD19- plasma cell population was detected, suggesting that downregulation of CD19 associated with maturation of plasma cells occurred. When purified human B cells were transplanted, those findings were not observed. Our results indicate that differentiation and maturation of human B cells and plasma cells can be investigated by transplantation of human PBMC into the spleen of NOD/SCID mice. The model will be useful for studying the differentiation of human B cells and generation of plasma cells.
Primary biliary cirrhosis (PBC) is characterized by an intense biliary inflammatory CD4+ and CD8+ T cell response. Very limited information on autoantigen-specific cytotoxic T lymphocyte (CTL) responses is available compared with autoreactive CD4+ T cell responses. Using peripheral blood mononuclear cells (PBMCs) from PBC, we identified an HLA-A2–restricted CTL epitope of the E2 component of pyruvate dehydrogenase (PDC-E2), the immunodominant mitochondrial autoantigen. This peptide, amino acids 159–167 of PDC-E2, induces specific MHC class I–restricted CD8+ CTL lines from 10/12 HLA-A2+ PBC patients, but not controls, after in vitro stimulation with antigen-pulsed dendritic cells (DCs). PDC-E2–specific CTLs could also be generated by pulsing DCs with full-length recombinant PDC-E2 protein. Furthermore, using soluble PDC-E2 complexed with either PDC-E2–specific human monoclonal antibody or affinity-purified autoantibodies against PDC-E2, the generation of PDC-E2–specific CTLs, occurred at 100-fold and 10-fold less concentration, respectively, compared with soluble antigen alone. Collectively, these data demonstrate that autoantibody, helper, and CTL epitopes all contain a shared peptide sequence. The finding that autoantigen–immune complexes can not only cross-present but also that presentation of the autoantigen is of a higher relative efficiency, for the first time defines a unique role for autoantibodies in the pathogenesis of an autoimmune disease.
autoimmunity; cytotoxic T cells; cholangitis; epitopes; cross-priming
While the pathologic mechanisms responsible for organ-specific tissue damage in primary biliary cirrhosis (PBC) remain an enigma, it has been suggested that the pathology is mediated by autoreactive T cells infiltrating the intrahepatic bile ducts. Previously, we have documented that there is 100-fold enrichment in the frequency of CD4+ autoreactive T cells in the liver that are specific for peptides encoded by the E2 components of the pyruvate dehydrogenase complexes (PDC-E2). We have also recently characterized the first MHC class I–restricted epitope for PDC-E2, namely amino acid 159–167, a region very similar to the epitope recognized by MHC class II–restricted CD4+ cells and by autoantibodies. The effector functions of these PDC-E2159-167–specific CD8+ cytotoxic T lymphocytes (CTLs) are not well understood. We have taken advantage of tetramer technology and report herein that there is tenfold increase in the frequency of PDC-E2159-167–specific CTLs in the liver as compared with the blood in PBC. In addition, the precursor frequency of the CTLs in blood was significantly higher in early-stage PBC. Of interest was the fact that, upon stimulation with the peptide, the response of PDC-E2159-167 tetramer-positive cells is heterogeneous with respect to IFN-γ synthesis. These data, we believe for the first time, document the enrichment of autoantigen-specific CD8+ T cells in the PBC liver, suggesting that CD8+ T cells play a significant role in the immunopathogenesis of PBC.
dnTGFβRII mice, expressing a dominant negative form of TGFβ receptor II under control of the CD4 promoter, develop autoimmune colitis and cholangitis . We previously observed that deficiency in IL-12p40 led to a marked diminution of inflammation in both the colon and the liver. To distinguish whether IL-12p40 mediated protection acted via the IL-12 or IL-23 pathways, we generated an IL-23p19−/− dnTGFβRII strain deficient in IL-23 but not in IL-12; mice were longitudinally followed for changes in the natural history of disease and immune responses. Interestingly, IL-23p19−/− mice demonstrate dramatic improvement in their colitis but no changes in biliary pathology; mice also manifest reduced Th17 cell populations and unchanged IFN-γ levels. We submit that the IL-12/Th1 pathway is essential for biliary disease pathogenesis, while the IL-23/Th17 pathway mediates colitis. To further assess the mechanism of the IL-23 mediated protection from colitis, we generated an IL-17A−/− dnTGFβRII strain deficient in IL-17, a major effector cytokine produced by IL-23-dependent Th17 cells. Deletion of the IL-17A gene did not affect the severity of either cholangitis or colitis, suggesting that the IL-23/Th17 pathway contributes to the colon disease in an IL-17-independent manner. These results affirm that the IL-12/Th1 pathway is critical to biliary pathology in dnTGFβRII mice while the colitis is caused by a direct effect of IL-23.
Primary biliary cirrhosis; murine models; autoimmunity; cholangitis; colitis
Hepatosplenic T cell lymphoma (HSTCL) is a distinct and lethal subtype of peripheral T cell lymphoma with an aggressive course and poor outcome despite multiagent chemotherapy. Contradictory literature, an unknown etiology, and poor response to treatment highlight the need to define the malignant process and identify molecular targets with potential for successful therapeutic interventions. Herein, we report that mice homozygously expressing a dominant negative TGFβRII (dnTGFβRII) under the control of the CD4 promoter spontaneously develop lymphoma-like T cell infiltration involving both spleen and liver. Splenomegaly, hepatomegaly and liver dysfunction were observed in homozygous dnTGFβRII mice between 10 weeks and 10 months of age associated with a predominant infiltration of CD4−CD8−TCRβ+NK1.1+ or CD8+TCRβ+NK1.1− T cell subsets. Notch 1 and c-Myc expression at the mRNA levels were significantly increased and positively correlated with the cell number of lymphoid infiltrates in the liver of dnTGFβRII homozygous compared to hemizygous mice. Further, 2×104 isolated lymphoma-like cells transplant disease by adoptive cell transfers. Collectively, our data demonstrate that increased copy number of dnTGFβRII is critical for development of lymphoma-like T cell infiltration.
In primary biliary cirrhosis (PBC), patients develop a multilineage response to a highly restricted peptide of the E2 component of pyruvate dehydrogenase (PDC-E2) involving autoantibody and autoreactive CD4+ and CD8+ T cell responses. Recent data from murine models have suggested that liver-infiltrating CD8+ cells play a critical role in biliary destruction in PBC. We hypothesized that chronic antigen stimulation of CD8+ T cells alters effector memory T cell (TEM) frequency and function similar to that seen with chronic viral infections including failure to terminally differentiate and relative resistance to apoptosis. We have rigorously phenotyped CD8+ T cell subpopulations from 132 subjects, including 76 patients with PBC and 56 controls, and report a higher frequency of TEM cells characterized as CD45ROhighCD57+CD8high but expressing the gut homing integrin α4β7 in PBMC of PBC. These CD8high TEM cells have reduced expression of annexin V after TCR stimulation. Consistent with a TEM phenotype, CD45ROhighCD57+CD8high T cells express higher levels of granzyme A, granzyme B, perforin, CCR5 and α4β7, and lower levels of CCR7 and CD28 than other CD8high T cells. Furthermore, interleukin (IL)-5 produced by CD8+CD57+ T lymphocytes upon in vitro TCR stimulation are increased in PBC. Histologically, CD8+CD57+ T cells accumulate around the portal area in PBC. Moreover, CD8+CD57+ T cells respond specifically to the MHC class I epitope of PDC-E2. In conclusion, our data demonstrate that CD45ROhighCD57+CD8high T cells are a subset of terminally differentiated cytotoxic TEM cells which could play a critical role in the progressive destruction of biliary epithelial cells.
Cytotoxic T cells; effector memory cells; cell sorting; homing receptors; annexin V