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1.  Clonality, Activated Antigen Specific CD8+ T Cells and Development of Autoimmune Cholangitis in dnTGFβRII Mice 
Hepatology (Baltimore, Md.)  2013;58(3):1094-1104.
There are several murine models described with features similar to human primary biliary cirrhosis (PBC). Amongst these models, the one which has the closest serologic features to PBC is a mouse with a T cell-restricted expression of the dominant negative transforming growth factor β receptor type II (dnTGFβRII). Our work has demonstrated that CD8+ T cells from dnTGFβRII mice transfer autoimmune cholangitis to Rag1-/- recipients. However, it remained unclear whether the autoimmune cholangitis was secondary to an intrinsic function within CD8+ T cells or due to the abnormal TGFβR environment within which CD8+ T cells were generated. To address this mechanistic issue, we utilized our dnTGFβRII, OT-I/Rag1-/-, OT-II/Rag1-/- mice and in addition generated OT-I/dnTGFβRII/Rag1-/-, and OT-II/dnTGFβRII/Rag1-/- mice in which the entire T cell repertoire was replaced with ovalbumin (OVA) specific CD8+ or CD4+ T cells, respectively. Importantly, neither the parental OT-I/dnTGFβRII/Rag1-/- mice and/or OT-II/dnTGFβRII/Rag1-/- mice developed cholangitis. However, adoptive transfer demonstrated that only transfer of CD8+ T cells from dnTGFβRII mice but not CD8+ T cells from OT-I/Rag -/- mice or from OT-I/dnTGFβRII/Rag1-/- mice transferred disease. These data were not secondary to absence of CD4+ T cell help since a combination of CD8+ T cells from OT-I/dnTGFβRII/Rag1-/- and CD4+ T cells from OT II/dnTGFβRII/Rag1-/- or CD8+ T cells from OT-I/dnTGFβRII/Rag1-/- with CD4+ T cells from OT-II/Rag1-/- mice failed to transfer disease. In conclusion, defective TGFβRII signaling, in addition to clonal CD8+ T cells that target biliary cells, are required for induction of autoimmune cholangitis.
doi:10.1002/hep.26418
PMCID: PMC3716874  PMID: 23532950
primary biliary cirrhosis; murine models; autoimmunity; cholangitis
2.  Heterovariant Cross-Reactive B-Cell Responses Induced by the 2009 Pandemic Influenza Virus A Subtype H1N1 Vaccine 
The Journal of Infectious Diseases  2012;207(2):288-296.
Background. The generation of heterovariant immunity is a highly desirable feature of influenza vaccines. The goal of this study was to compare the heterovariant B-cell response induced by the monovalent inactivated 2009 pandemic influenza A virus subtype H1N1 (A[H1N1]pdm09) vaccine with that induced by the 2009 seasonal trivalent influenza vaccine (sTIV) containing a seasonal influenza A virus subtype H1N1 (A[H1N1]) component in young and elderly adults.
Methods. Plasmablast-derived polyclonal antibodies (PPAb) from young and elderly recipients of A(H1N1)pdm09 vaccine or sTIV were tested for binding activity to various influenza antigens.
Results. In A(H1N1)pdm09 recipients, the PPAb titers against homotypic A(H1N1)pdm09 vaccine were similar to those against the heterovariant seasonal A(H1N1) vaccine and were similar between young and elderly subjects. The PPAb avidity was higher among elderly individuals, compared with young individuals. In contrast, the young sTIV recipients had 10-fold lower heterovariant PPAb titers against the A(H1N1)pdm09 vaccine than against the homotypic seasonal A(H1N1) vaccine. In binding assays with recombinant head and stalk domains of hemagglutinin, PPAb from the A(H1N1)pdm09 recipients but not PPAb from the sTIV recipients bound to the conserved stalk domain.
Conclusion. The A(H1N1)pdm09 vaccine induced production of PPAb with heterovariant reactivity, including antibodies targeting the conserved hemagglutinin stalk domain.
doi:10.1093/infdis/jis664
PMCID: PMC3532823  PMID: 23107783
influenza; vaccine; antibody; cross-reactivity
3.  Plasma Cells and the Chronic Nonsuppurative Destructive Cholangitis of Primary Biliary Cirrhosis 
Hepatology (Baltimore, Md.)  2012;55(3):846-855.
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.
doi:10.1002/hep.24757
PMCID: PMC3272098  PMID: 22031474
antimitochondrial antibodies; primary biliary cirrhosis; plasma cells; coronal arrangement
4.  Epithelial Cell Specificity and Apotope Recognition by Serum Autoantibodies in Primary Biliary Cirrhosis 
Hepatology (Baltimore, Md.)  2011;54(1):196-203.
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.
doi:10.1002/hep.24355
PMCID: PMC3125447  PMID: 21488079
Apoptosis; human intrahepatic biliary epithelial cells; E2 subunit of 2-oxoacid dehydrogenase complex; nuclear antigens; autoimmunity
5.  Plasmablast-derived polyclonal antibody response after influenza vaccination 
Journal of immunological methods  2010;365(1-2):67-75.
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.
doi:10.1016/j.jim.2010.12.008
PMCID: PMC3039424  PMID: 21182843
Influenza virus; vaccines; antibodies; plasmablasts
6.  Limited efficacy of inactivated influenza vaccine in elderly individuals is associated with decreased production of vaccine-specific antibodies 
The Journal of Clinical Investigation  2011;121(8):3109-3119.
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.
doi:10.1172/JCI57834
PMCID: PMC3148747  PMID: 21785218
7.  Differential Transcriptional Responses to Interferon-α and Interferon-γ in Primary Human Hepatocytes 
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.
doi:10.1089/jir.2009.0082
PMCID: PMC2947462  PMID: 20038212
8.  Deficiency in regulatory T cells results in development of antimitochondrial antibodies and autoimmune cholangitis 
Hepatology (Baltimore, Md.)  2009;49(2):545-552.
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.
doi:10.1002/hep.22651
PMCID: PMC2635418  PMID: 19065675
Sf mouse; Primary Biliary Cirrhosis; Tregs; Interleukin 12; CD8 T lymphocyte
9.  T cell–dependent production of IFN-γ by NK cells in response to influenza A virus 
Journal of Clinical Investigation  2004;114(12):1812-1819.
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.
doi:10.1172/JCI200422797
PMCID: PMC535070  PMID: 15599406
10.  Overexpression of microRNA-21 is associated with elevated pro-inflammatory cytokines in dominant-negative TGF-β receptor type II mouse 
Journal of autoimmunity  2013;41:111-119.
Dominant negative TGF-β receptor II (dnTGF-βRII) mice spontaneously develop an autoimmune cholangitis resembling human primary biliary cirrhosis (PBC). Interestingly, the dominant negative TGF-β receptor is expressed by both CD4+ and CD8+ T cells and leads to greatly reduced (but not absent) TGF-β signaling resulting in T cell intrinsic cell mediated autoimmunity. However, the mechanisms of the T cell dysregulation remain unclear. Recently it has been shown that TGF-β signaling is intimately involved with miRNA biogenesis and control. Herein we show that lack of T cell TGF-β signaling leads to down regulation of T cell miRNAs but upregulation of the key inflammatory miRNA 21. Furthermore, the expression of miR-21 from hepatic effector CD8+ T cells is significantly higher than in the same subsets isolated from spleen and mesenteric lymph nodes of the dnTGF-βRII mice. Previous studies indicate that miR-21 increases the synthesis of IFN-γ and IL-17A by T cells and suppresses apoptosis via programmed cell death protein 4 (PDCD4). Data presented herein demonstrate that transfecting w.t. B6 T cell subsets with miR-21 resulted in upregulation of the inflammatory cytokines TNF-α and IFN-γ, thus partly replicating the dnTGF-βRII T cell phenotype. In conclusion, these data suggest miR-21 plays a critical role in the production of pro-inflammatory cytokines in dnTGFβRII mice, which could be a contributing factor for the development of the organ-specific autoimmune cholangitis and colitis in this murine model of human PBC.
doi:10.1016/j.jaut.2012.12.013
PMCID: PMC3622842  PMID: 23395552
Primary biliary cirrhosis; inflammatory bowel disease; autoimmunity; cholangitis; colitis; microRNA; miR-21
11.  Deletion of IL-12p35 induces liver fibrosis in dominant negative transforming growth factor β receptor type II mice 
Hepatology (Baltimore, Md.)  2013;57(2):806-816.
We have previously reported that mice with a dominant negative transforming growth factor β receptor restricted to T cells (dnTGFβRII mice) develop an inflammatory biliary ductular disease that strongly resembles human primary biliary cirrhosis (PBC). Furthermore, deletion of the gene encoding interleukin (IL)-12p40 resulted in a strain (IL-12p40−/−dnTGFβRII) with dramatically reduced autoimmune cholangitis. To further investigate the role of the IL-12 cytokine family in dnTGFβRII autoimmune biliary disease, we deleted the gene encoding the IL-12p35 subunit from dnTGFβRII mice, resulting in an IL-12p35−/− dnTGFβRII strain which is deficient in two members of the IL-12 family, IL-12 and IL-35. In contrast to IL-12p40−/− mice, the IL-12p35−/− mice developed liver inflammation and bile duct damage with similar severity but delayed onset as the parental dnTGFβRII mice. The p35−/− mice also demonstrated a distinct cytokine profile characterized by a shift from a Th1 to a Th17 response. Strikingly, liver fibrosis was frequently observed in IL-12p35−/− mice. In conclusion, IL-12p35−/− dnTGFβRII mice, histologically and immunologically, reflect key features of PBC, providing a useful generic model to understand the immunopathology of human PBC.
doi:10.1002/hep.25829
PMCID: PMC3424295  PMID: 22576253
Primary biliary cirrhosis; murine models; autoimmunity; cholangitis
12.  Therapeutic effect of CTLA4-Ig on a murine model of primary biliary cirrhosis 
Hepatology (Baltimore, Md.)  2013;57(2):708-715.
Collectively, the data in both humans and murine models of human primary biliary cirrhosis (PBC) suggest that activated T cells, particularly CD8 T cells, play a critical role in biliary cell destruction. Under physiological conditions, T cell activation involves two critical signals that involve the MHC and a set of co-stimulatory molecules which include a receptor on T cells coined cytotoxic T lymphocyte antigen 4 (CTLA-4). Germane to the studies reported herein, signaling via CTLA-4 has the potential to modulate co-stimulation and induce inhibitory signals. In this study we have taken advantage of our well-defined murine model of PBC in which mice are immunized with 2-octynoic acid coupled to BSA, leading to the production of high titer anti-mitochondrial autoantibodies and portal cellular infiltrates. To investigate the potential of CTLA-4 Ig as an immunotherapeutic agent, we treated mice both before and after induction of autoimmune cholangitis. Firstly, we demonstrate that CTLA-4 Ig treatment begun one day before 2-OA-BSA immunization, completely inhibits the manifestations of cholangitis, including AMA production, intra-hepatic T cell infiltrates and bile duct damage. However, and more critically, treatment with CTLA-4 Ig initiated after the development of autoimmune cholangitis in previously immunized mice, also resulted in significant therapeutic benefit, including reduced intra-hepatic T cell infiltrates and biliary cell damage, although AMA levels were not altered. These data suggest that an optimized regimen with CTLA-4 Ig has the potential to serve as an investigative therapeutic tool in patients with PBC.
doi:10.1002/hep.26067
PMCID: PMC3548049  PMID: 22996325
Primary biliary cirrhosis; cytotoxic T lymphocyte antigen 4; Abatacept autoimmunity; cholangitis
13.  The Immunobiology of Colitis and Cholangitis in IL-23p19 and IL-17A Deleted dnTGFβRII Mice 
Hepatology (Baltimore, Md.)  2012;56(4):1418-1426.
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.
doi:10.1002/hep.25803
PMCID: PMC3415593  PMID: 22532156
Primary biliary cirrhosis; murine models; autoimmunity; cholangitis; colitis
14.  Identification of Potential Cytokine Pathways for Therapeutic Intervention in Murine Primary Biliary Cirrhosis 
PLoS ONE  2013;8(9):e74225.
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.
doi:10.1371/journal.pone.0074225
PMCID: PMC3769355  PMID: 24040208
15.  Lineage Structure of the Human Antibody Repertoire in Response to Influenza Vaccination 
Science translational medicine  2013;5(171):171ra19.
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.
doi:10.1126/scitranslmed.3004794
PMCID: PMC3699344  PMID: 23390249
16.  Lymphoma-Like T Cell Infiltration in Liver Is Associated with Increased Copy Number of Dominant Negative Form of TGFβ Receptor II 
PLoS ONE  2012;7(11):e49413.
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.
doi:10.1371/journal.pone.0049413
PMCID: PMC3492285  PMID: 23145171
17.  Fine phenotypic and functional characterization of effector CD8+ T cells in patients with Primary Biliary Cirrhosis 
Hepatology (Baltimore, Md.)  2011;54(4):1293-1302.
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.
doi:10.1002/hep.24526
PMCID: PMC3184190  PMID: 21735469
Cytotoxic T cells; effector memory cells; cell sorting; homing receptors; annexin V
18.  Rejection of Unfair Offers Can Be Driven by Negative Emotions, Evidence from Modified Ultimatum Games with Anonymity 
PLoS ONE  2012;7(6):e39619.
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.
doi:10.1371/journal.pone.0039619
PMCID: PMC3386255  PMID: 22761845
19.  The Influence of CD4+ CD25+ Foxp3+ Regulatory T Cells on the Immune Response to Rotavirus Infection 
Vaccine  2008;26(44):5601-5611.
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.
doi:10.1016/j.vaccine.2008.07.099
PMCID: PMC2574936  PMID: 18725261
Rotavirus; Regulatory T cell; vaccines
20.  Qualitative and Quantitative Characteristics of Rotavirus-Specific CD8 T Cells Vary Depending on the Route of Infection▿  
Journal of Virology  2008;82(14):6812-6819.
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.
doi:10.1128/JVI.00450-08
PMCID: PMC2446946  PMID: 18480435
21.  Influence of Prior Influenza Vaccination on Antibody and B-Cell Responses 
PLoS ONE  2008;3(8):e2975.
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.
doi:10.1371/journal.pone.0002975
PMCID: PMC2500171  PMID: 18714352
22.  Baseline Levels of Influenza-Specific CD4 Memory T-Cells Affect T-Cell Responses to Influenza Vaccines 
PLoS ONE  2008;3(7):e2574.
Background
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.
Methodology/Principal Findings
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.
Significance
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.
doi:10.1371/journal.pone.0002574
PMCID: PMC2440350  PMID: 18596908
23.  Cellular Immune Responses in Children and Adults Receiving Inactivated or Live Attenuated Influenza Vaccines▿  
Journal of Virology  2006;80(23):11756-11766.
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.
doi:10.1128/JVI.01460-06
PMCID: PMC1642596  PMID: 16971435
24.  Appearance of Human Plasma Cells Following Differentiation of Human B Cells in NOD/SCID Mouse Spleen 
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.
doi:10.1080/10446670310001642122
PMCID: PMC2485411  PMID: 14768952
25.  Identification of HLA-A2–restricted CD8+ Cytotoxic T Cell Responses in Primary Biliary Cirrhosis 
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.
doi:10.1084/jem.20010956
PMCID: PMC2196012  PMID: 11781370
autoimmunity; cytotoxic T cells; cholangitis; epitopes; cross-priming

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