Jinguo Wang

Reversal of Autoimmunity by Boosting Memory-like Autoregulatory T Cells

Blunting autoreactivity without compromising immunity remains an elusive goal in the treatment of autoimmunity. We show that progression to autoimmune diabetes results in the conversion of naive low-avidity autoreactive CD8(+) T cells into memory-like autoregulatory cells that can be expanded in vivo with nanoparticles coated with disease-relevant peptide-major histocompatibility complexes (pMHC-NP). Treatment of NOD mice with monospecific pMHC-NPs expanded cognate autoregulatory T cells, suppressed the recruitment of noncognate specificities, prevented disease in prediabetic mice, and restored normoglycemia in diabetic animals. pMHC-NP therapy was inconsequential in mice engineered to bear an immune system unresponsive to the corresponding epitope, owing to absence of epitope-experienced autoregulatory T cells. pMHC-NP-expanded autoregulatory T cells suppressed local presentation of autoantigens in an interferon-gamma-, indoleamine 2,3-dioxygenase-, and perforin-dependent manner. Nanoparticles coated with human diabetes-relevant pHLA complexes restored normoglycemia in a humanized model of diabetes. These observations expose a paradigm in the pathogenesis of autoimmunity amenable for therapeutic intervention.

Expanding antigen-specific regulatory networks to treat autoimmunity

Regulatory T cells hold promise as targets for therapeutic intervention in autoimmunity, but approaches capable of expanding antigen-specific regulatory T cells in vivo are currently not available. Here we show that systemic delivery of nanoparticles coated with autoimmune-disease-relevant peptides bound to major histocompatibility complex class II (pMHCII) molecules triggers the generation and expansion of antigen-specific regulatory CD4+ T cell type 1 (TR1)-like cells in different mouse models, including mice humanized with lymphocytes from patients, leading to resolution of established autoimmune phenomena. Ten pMHCII-based nanomedicines show similar biological effects, regardless of genetic background, prevalence of the cognate T-cell population or MHC restriction. These nanomedicines promote the differentiation of disease-primed autoreactive T cells into TR1-like cells, which in turn suppress autoantigen-loaded antigen-presenting cells and drive the differentiation of cognate B cells into disease-suppressing regulatory B cells, without compromising systemic immunity. pMHCII-based nanomedicines thus represent a new class of drugs, potentially useful for treating a broad spectrum of autoimmune conditions in a disease-specific manner.

IL-2 and its high-affinity receptor: Genetic control of immunoregulation and autoimmunity

Type 1 diabetes (T1D) is an organ-specific autoimmune disease featured by destruction of the insulin producing beta-cells of the pancreas by autoreactive T-lymphocytes. Putative environmental triggers conspire with a constellation of genetic elements scattered throughout the genome to elicit a multifactorial autoimmune response involving virtually every cell type of the immune system against pancreatic beta-cells. Recent highly powered genome-wide association studies have confirmed and identified fifteen chromosomal regions harboring several candidate T1D-associated gene loci. Here, we summarize what we know about the genetics of T1D with an emphasis on the contributions of mouse Il2 and human IL2RA polymorphisms and the IL-2-IL-2R pathway to autoimmunity and, more specifically, Treg development and function.

The Proline-Rich Sequence of CD3ε as an Amplifier of Low-Avidity TCR Signaling

Engagement of peptide-MHC by the TCR induces a conformational change in CD3ε that exposes a proline-rich sequence (PRS) and recruits the cytoskeletal adaptor Nck. This event, which precedes phosphorylation of the CD3ε ITAM, has been implicated in synapse formation and T cell function. However, there is compelling evidence that responsiveness to TCR ligation is CD3ε PRS independent. In this study, we show that the CD3ε PRS is necessary for peptide-MHC-induced phosphorylation of CD3ε and for recruitment of protein kinase Cθ to the immune synapse in differentiated CD8+ T lymphocytes. However, whereas these two events are dispensable for functional T cell responsiveness to high-avidity ligands, they are required for responsiveness to low-avidity ones. Thus, in at least certain T cell clonotypes, the CD3ε PRS amplifies weak TCR signals by promoting synapse formation and CD3ε phosphorylation.

In situ recognition of autoantigen as an essential gatekeeper in autoimmune CD8+ T cell inflammation

A current paradigm states that non-antigen-specific inflammatory cues attract noncognate, bystander T cell specificities to sites of infection and autoimmune inflammation. Here we show that cues emanating from a tissue undergoing spontaneous autoimmune inflammation cannot recruit naive or activated bystander T cell specificities in the absence of local expression of cognate antigen. We monitored the recruitment of CD8+ T cells specific for the prevalent diabetogenic epitope islet-specific glucose-6-phosphatase catalytic subunit-related protein (IGRP)206–214 in gene-targeted nonobese diabetic (NOD) mice expressing a T cell “invisible” IGRP206–214 sequence. These mice developed islet inflammation and diabetes with normal incidence and kinetics, but their inflammatory lesions could recruit neither naive (endogenous or exogenous) nor ex vivo-activated IGRP206–214-reactive CD8+ T cells. Conversely, IGRP206–214-reactive, but not nonautoreactive CD8+ T cells rapidly homed to and accumulated in the inflamed islets of wild-type NOD mice. Our results indicate that CD8+ T cell recruitment to a site of autoimmune inflammation results from an active process that is strictly dependent on local display of cognate pMHC and suggest that CD8+ T cells contained in extralymphoid autoimmune lesions are largely autoreactive.

Bicistronic Woodchuck Hepatitis Virus Core and Gamma Interferon DNA Vaccine Can Protect from Hepatitis but Does Not Elicit Sterilizing Antiviral Immunity

ABSTRACT The immunity elicited against nucleocapsid of hepatitis B virus (HBV) and closely related woodchuck hepatitis virus (WHV) has been shown to be important in resolution of hepatitis and protection from infection. Further, activity of gamma interferon (IFN-γ), which may directly inhibit hepadnavirus replication, promotes antiviral defense and favors T helper cell type 1 (Th1) response, which is seemingly a prerequisite of HBV clearance. In this study, to enhance induction of protective immunity against hepadnavirus, healthy woodchucks were immunized with a bicistronic DNA vaccine carrying WHV core (WHc) and woodchuck IFN-γ (wIFN-γ) gene sequences. Three groups, each group containing three animals, were injected once or twice with 0.5 mg, 0.9 mg, or 1.5 mg per dose of this vaccine. In addition, four animals received two injections of 0.6 mg or 1 mg WHc DNA alone. All animals were challenged with WHV. The results showed that four of nine animals injected with the bicistronic vaccine and one of four immunized with WHc DNA became protected from serologically evident infection and hepatitis. This protection was not linked to induction of WHc antigen-specific antibodies or T-cell proliferative response and was not associated with enhanced transcription of Th1 cytokines or 2′,5′-oligoadenylate synthetase. Strikingly, all animals protected from hepatitis became reactive for WHV DNA and carried low levels of replicating virus in hepatic and lymphoid tissues after challenge with WHV. This study shows that the bicistronic DNA vaccine encoding both hepadnavirus core antigen and IFN-γ was more effective in preventing hepatitis than that encoding virus core alone, but neither of them could mount sterile immunity against the virus or prevent establishment of occult infection.

Hepatocytes as cytotoxic effector cells can induce cell death by CD95 ligand-mediated pathway†

The liver plays an increasingly recognized role in the hosts immune responses. The direct contribution of hepatocytes as effector cells to local immunity, pathogen containment, and liver disease is not determined. This in vitro study examined whether hepatocytes can eliminate other cells via a CD95 ligand (CD95L or FasL)/CD95 (Fas)–mediated mechanism and whether this cytotoxic activity can be modulated by cytokines such as interferon gamma (IFN‐γ) or tumor necrosis factor alpha (TNF‐α). We have found that normal woodchuck and human hepatocytes, both cultured and primary freshly isolated, as well as human HepG2 cells, intrinsically transcribe not only CD95 but also CD95L when examined by reverse transcription‐polymerase chain reaction (RT‐PCR) assays. The functional competence of CD95L, which was detectable in hepatocytes and HepG2 cells by Western blotting, was confirmed in bioassays by induction of apoptosis of CD95‐bearing P815 and LS102.9 cell targets and validated by inhibition of the cell killing with CD95 antagonistic antibody or with a general caspase inhibitor. Furthermore, exposure of cultured hepatocytes to IFN‐γ or their stable transfection with IFN‐γ cDNA or TNF‐α cDNA increased hepatocyte CD95L/CD95–mediated cell killing. In conclusion, hepatocytes express both CD95L and CD95 and they can induce death of other cells by a CD95L‐dependent mechanism. IFN‐γ and, to a lesser extent, TNF‐α can enhance hepatocyte CD95L‐mediated cytotoxicity. This suggests that the local cytokine environment may modulate the hepatocyte contribution to liver immunity. (HEPATOLOGY 2006;43:1231–1240.)

Inhibition by Woodchuck Hepatitis Virus of Class I Major Histocompatibility Complex Presentation on Hepatocytes Is Mediated by Virus Envelope Pre-S2 Protein and Can Be Reversed by Treatment with Gamma Interferon

ABSTRACT Presentation of class I major histocompatibility complex (MHC) is severely down-regulated on hepatocytes in chronic hepatitis caused by woodchuck hepatitis virus (WHV). To determine which of the viral proteins mediates class I MHC antigen suppression, cultured normal woodchuck hepatocytes were transfected with the complete WHV genome, sequences encoding individual virus proteins, or whole virus genomes in which transcription of selected proteins was disabled by site-specific mutagenesis. It was found that hepatocyte presentation of class I MHC antigen was significantly inhibited following transfection with complete WHV genome or with viral subgenomic fragments encoding envelope pre-S2 protein or pre-S1 protein, which naturally encompasses pre-S2 amino acid sequence. In contrast, hepatocytes transfected with WHV X gene alone demonstrated a profound enhancement in the class I antigen display, whereas those expressing virus major S protein or nucleocapsid (core) protein were not different from control hepatocytes. Analysis of the mutated WHV sequences confirmed that the envelope pre-S2 protein was responsible for inhibition of the class I MHC antigen display. Interestingly, treatment with recombinant woodchuck gamma interferon (rwIFN-γ) restored the inhibited presentation of the class I antigen. Moreover, the class I antigen suppression was not associated with down-regulation of hepatocyte genes for class I MHC heavy chain, β2-microglobulin, transporters associated with antigen processing, and proteasome subunits. These findings indicate that the defective presentation of class I MHC antigen on hepatocytes transcribing WHV is a consequence of posttranscriptional suppression exerted by virus pre-S2 protein and that this hindrance can be fully reversed by IFN-γ.

Development of Memory-Like Autoregulatory CD8+ T Cells Is CD4+ T Cell Dependent

Progression of spontaneous autoimmune diabetes is associated with development of a disease-countering negative-feedback regulatory loop that involves differentiation of low-avidity autoreactive CD8+ cells into memory-like autoregulatory T cells. Such T cells blunt diabetes progression by suppressing the presentation of both cognate and noncognate Ags to pathogenic high-avidity autoreactive CD8+ T cells in the pancreas-draining lymph nodes. In this study, we show that development of autoregulatory CD8+ T cell memory is CD4+ T cell dependent. Transgenic (TG) NOD mice expressing a low-affinity autoreactive TCR were completely resistant to autoimmune diabetes, even after systemic treatment of the mice with agonistic anti-CD40 or anti–4-1BB mAbs or autoantigen-pulsed dendritic cells, strategies that dramatically accelerate diabetes development in TG NOD mice expressing a higher affinity TCR for the same autoantigenic specificity. Furthermore, whereas abrogation of RAG-2 expression, hence endogenous CD4+ T cell and B cell development, decelerated disease progression in high-affinity TCR-TG NOD mice, it converted the low-affinity TCR into a pathogenic one. In agreement with these data, polyclonal CD4+ T cells from prediabetic NOD mice promoted disease in high-affinity TCR-TG NOD.Rag2−/− mice, but inhibited it in low-affinity TCR-TG NOD.Rag2−/− mice. Thus, in chronic autoimmune responses, CD4+ Th cells contribute to both promoting and suppressing pathogenic autoimmunity.

Hepatocytes can induce death of contacted cells via perforin-dependent mechanism†

The liver displays unique immunological properties including the ability to remove aberrant cells and pathogens and to induce peripheral immunotolerance. We have previously demonstrated that hepatocytes can cause cell death by a CD95 ligand–mediated mechanism. Here, we provide evidence that hepatocytes can kill other cells via a perforin‐dependent pathway. Using cultured woodchuck hepatocytes and human liver cells as well as freshly isolated woodchuck, mouse, and human hepatocytes, we show that hepatocyte‐mediated death of CD95‐deficient target cells requires microtubule polymerization, a feature of the granule exocytosis–mediated cytotoxicity. Neutralizing anti‐perforin antibodies and short‐hairpin RNA directed against perforin messenger RNA confirmed the involvement of perforin in hepatocyte‐mediated cell killing. Conclusion: This study shows that hepatocytes express biologically competent perforin capable of killing susceptible cells and emphasizes the role of hepatocytes as cytotoxic effectors. This also is the first demonstration of perforin in a non‐lymphoid cell type. (HEPATOLOGY 2008.)