The programmed death-1 (PD-1) pathway regulates autoimmune diabetes in nonobese diabetic (NOD) mice

Programmed death-1 (PD-1) receptor, an inhibitory costimulatory molecule found on activated T cells, has been demonstrated to play a role in the regulation of immune responses and peripheral tolerance. We investigated the role of this pathway in the development of autoimmune diabetes. PD-1 or PD-L1 but not PD-L2 blockade rapidly precipitated diabetes in prediabetic female nonobese diabetic (NOD) mice regardless of age (from 1 to 10-wk-old), although it was most pronounced in the older mice. By contrast, cytotoxic T lymphocyte-associated antigen 4 (CTLA-4) blockade induced disease only in neonates. Male NOD mice also developed diabetes after PD-1-PD-L1 pathway blockade, but NOR mice, congenic to NOD but resistant to the development of diabetes, did not. Insulitis scores were significantly higher and frequency of interferon gamma-producing GAD-reactive splenocytes was increased after PD-1-PD-L1 pathway blockade compared with controls. Interestingly, PD-L1 but not PD-L2 was found to be expressed on inflamed islets of NOD mice. These data demonstrate a central role for PD-1-PD-L1 interaction in the regulation of induction and progression of autoimmune diabetes in the NOD mouse and provide the rationale to develop new therapies to target this costimulatory pathway in this disease.     

Tissue expression of PD-L1 mediates peripheral T cell tolerance

Programmed death 1 (PD-1), an inhibitory receptor expressed on activated lymphocytes, regulates tolerance and autoimmunity. PD-1 has two ligands: PD-1 ligand 1 (PD-L1), which is expressed broadly on hematopoietic and parenchymal cells, including pancreatic islet cells; and PD-L2, which is restricted to macrophages and dendritic cells. To investigate whether PD-L1 and PD-L2 have synergistic or unique roles in regulating T cell activation and tolerance, we generated mice lacking PD-L1 and PD-L2 (PD-L1/PD-L2(-/-) mice) and compared them to mice lacking either PD-L. PD-L1 and PD-L2 have overlapping functions in inhibiting interleukin-2 and interferon-gamma production during T cell activation. However, PD-L1 has a unique and critical role in controlling self-reactive T cells in the pancreas. Our studies with bone marrow chimeras demonstrate that PD-L1/PD-L2 expression only on antigen-presenting cells is insufficient to prevent the early onset diabetes that develops in PD-L1/PD-L2(-/-) non-obese diabetic mice. PD-L1 expression in islets protects against immunopathology after transplantation of syngeneic islets into diabetic recipients. PD-L1 inhibits pathogenic self-reactive CD4+ T cell-mediated tissue destruction and effector cytokine production. These data provide evidence that PD-L1 expression on parenchymal cells rather than hematopoietic cells protects against autoimmune diabetes and point to a novel role for PD-1-PD-L1 interactions in mediating tissue tolerance.     

Role of Interleukin 12 and Costimulators in T Cell Anergy In Vivo

The induction of T cell anergy in vivo is thought to result from antigen recognition in the absence of co-stimulation and inflammation, and is associated with a block in T cell proliferation and Th1 differentiation. Here we have examined the role of interleukin (IL)-12, a potent inducer of Th1 responses, in regulating this process. T cell tolerance was induced by the administration of protein antigen without adjuvant in normal mice, and in recipients of adoptively transferred T cells from T cell receptor transgenic mice. The administration of IL-12 at the time of tolerance induction stimulates Th1 differentiation, but does not promote antigen-specific T cell proliferation. Conversely, inhibiting CTLA-4 engagement during anergy induction reverses the block in T cell proliferation, but does not promote full Th1 differentiation. T cells exposed to tolerogenic antigen in the presence of both IL-12 and anti–CTLA-4 antibody are not anergized, and behave identically to T cells which have encountered immunogenic antigen. These results suggest that two processes contribute to the induction of anergy in vivo; CTLA-4 engagement, which leads to a block in the ability of T cells to proliferate to antigen, and the absence of a prototypic inflammatory cytokine, IL-12, which prevents the differentiation of T cells into Th1 effector cells. The combination of IL-12 and anti–CTLA-4 antibody is sufficient to convert a normally tolerogenic stimulus to an immunogenic one.     

PD-1 inhibits antiviral immunity at the effector phase in the liver

Unlike naive T cells, effector T cells can be activated by either T cell receptor signal or costimulatory signal alone and therefore the absence of costimulatory molecules on tissue cells cannot explain the tolerance mechanism at the effector phase. Here we report that PD-L1, the ligand for the immunoinhibitory receptor PD-1, was expressed on vascular endothelium in peripheral tissues. Liver nonparenchymal cells including sinusoidal endothelial cells and Kupffer cells constitutively expressed PD-L1 and inhibited proliferation and cell division of activated T cells expressing PD-1. The absence of PD-1 induced proliferation of effector T cells in the adenovirus-infected liver and resulted in rapid clearance of the virus. These results indicate that PD-1 plays an important role in T cell tolerance at the effector phase and the blockade of the PD-1 pathway can augment antiviral immunity.     

PD-1/PD-L1 Blockade Can Enhance HIV-1 Gag-specific T Cell Immunity Elicited by Dendritic Cell-Directed Lentiviral Vaccines

Exhaustion of CD8⁺ T cells and upregulation of programmed death 1 (PD-1), a negative regulator of T cell activation, are characteristic features of individuals chronically infected with human immunodeficiency virus type 1. In a previous study, we showed in mice that a dendritic cell-directed lentiviral vector (DCLV) system encoding the human immunodeficiency virus (HIV)-1 Gag protein was an efficient vaccine modality to induce a durable Gag-specific T cell immune response. In this study, we demonstrate that blocking of the PD-1/PD-1 ligand (PD-L) inhibitory signal via an anti-PD-L1 antibody generated an enhanced HIV-1 Gag-specific CD8⁺ immune response following both a single round of DCLV immunization and a homologous prime/boost regimen. The prime/boost regimen combined with PD-L1 blockade generated very high levels of Gag-specific CD8⁺ T cells comprising several valuable features: improved ability to produce multiple cytokines, responding to a broader range of Gag-derived epitopes, and long-lasting memory. This enhanced cellular immune response generated by DCLV immunization combined with anti-PD-L1 blockade correlated with improved viral control following challenge with Gag-expressing vaccinia virus. Taken together, our studies offer evidence to support the use of PD-1/PD-L1 blockade as an adjuvant modality to enhance antigen-specific immune responses elicited by T cell-based immunizations such as DCLV.     

Critical role of the programmed death-1 (PD-1) pathway in regulation of experimental autoimmune encephalomyelitis

Experimental autoimmune encephalomyelitis (EAE) is mediated by autoantigen-specific T cells dependent on critical costimulatory signals for their full activation and regulation. We report that the programmed death-1 (PD-1) costimulatory pathway plays a critical role in regulating peripheral tolerance in murine EAE and appears to be a major contributor to the resistance of disease induction in CD28-deficient mice. After immunization with myelin oligodendrocyte glycoprotein (MOG) there was a progressive increase in expression of PD-1 and its ligand PD-L1 but not PD-L2 within the central nervous system (CNS) of mice with EAE, peaking after 3 wk. In both wild-type (WT) and CD28-deficient mice, PD-1 blockade resulted in accelerated and more severe disease with increased CNS lymphocyte infiltration. Worsening of disease after PD-1 blockade was associated with a heightened autoimmune response to MOG, manifested by increased frequency of interferon gamma-producing T cells, increased delayed-type hypersensitivity responses, and higher serum levels of anti-MOG antibody. In vivo blockade of PD-1 resulted in increased antigen-specific T cell expansion, activation, and cytokine production. Interestingly, PD-L2 but not PD-L1 blockade in WT animals also resulted in disease augmentation. Our data are the first demonstration that the PD-1 pathway plays a critical role in regulating EAE.     

Blockade ofthe negative co-stimulatory molecules PD-1 and CTLA-4 improves survival in primary and secondary fungal sepsis

Introduction

Fungal sepsis is an increasingly common problem in intensive care unit patients.Mortality from fungal sepsis remains high despite antimicrobial therapy that is highly active against most fungal pathogens, a finding consistent with defective host immunity that is present in many patients with disseminated fungemia.One recently recognized immunologic defect that occurs in patients with sepsis is T cell "exhaustion" due to increased expression of programmed cell death -1 (PD-1).This study tested the ability of anti-PD-1 and anti-programmed cell death ligand -1 (anti-PD-L1) antagonistic antibodies to improve survival and reverse sepsis-induced immunosuppression in two mouse models of fungal sepsis.

Methods

Fungal sepsis was induced in mice using two different models of infection, that is, primary fungal sepsis and secondary fungal sepsis occurring after sub-lethal cecal ligation and puncture (CLP).Anti-PD-1 and anti-PD-L1 were administered 24 to 48 h after fungal infection and effects on survival, interferon gamma production, and MHC II expression were examined.

Results

Anti-PD-1 and anti-PD-L1 antibodies were highly effective at improving survival in primary and secondary fungal sepsis.Both antibodies reversed sepsis-induced suppression of interferon gamma and increased expression of MHC II on antigen presenting cells.Blockade of cytotoxic T-lymphocyte antigen-4 (CTLA-4), a second negative co-stimulatory molecule that is up-regulated in sepsis and acts like PD-1 to suppress T cell function, also improved survival in fungal sepsis.

Conclusions

Immuno-adjuvant therapy with anti-PD-1, anti-PD-L1 and anti-CTLA-4 antibodies reverse sepsis-induced immunosuppression and improve survival in fungal sepsis.The present results are consistent with previous studies showing that blockade of PD-1 and CTLA-4 improves survival in bacterial sepsis.Thus, immuno-adjuvant therapy represents a novel approach to sepsis and may have broad applicability in the disorder.Given the relative safety of anti-PD-1 antibody in cancer clinical trials to date, therapy with anti-PD-1 in patients with life-threatening sepsis who have demonstrable immunosuppression should be strongly considered.     

PD-1 is a regulator of virus-specific CD8+ T cell survival in HIV infection

Here, we report on the expression of programmed death (PD)-1 on human virus-specific CD8(+) T cells and the effect of manipulating signaling through PD-1 on the survival, proliferation, and cytokine function of these cells. PD-1 expression was found to be low on naive CD8(+) T cells and increased on memory CD8(+) T cells according to antigen specificity. Memory CD8(+) T cells specific for poorly controlled chronic persistent virus (HIV) more frequently expressed PD-1 than memory CD8(+) T cells specific for well-controlled persistent virus (cytomegalovirus) or acute (vaccinia) viruses. PD-1 expression was independent of maturational markers on memory CD8(+) T cells and was not directly associated with an inability to produce cytokines. Importantly, the level of PD-1 surface expression was the primary determinant of apoptosis sensitivity of virus-specific CD8(+) T cells. Manipulation of PD-1 led to changes in the ability of the cells to survive and expand, which, over several days, affected the number of cells expressing cytokines. Therefore, PD-1 is a major regulator of apoptosis that can impact the frequency of antiviral T cells in chronic infections such as HIV, and could be manipulated to improve HIV-specific CD8(+) T cell numbers, but possibly not all functions in vivo.     

siRNA-mediated silencing of PD-1 ligands enhances tumor-specific human T-cell effector functions

Adoptive cell therapy using tumor-specific T cells is a promising strategy for treating patients with malignancy. However, accumulating evidences have demonstrated that optimal function of tumor-reactive T cells is often attenuated by negative regulatory signal(s) delivered through receptors, such as cytotoxic T-lymphocyte antigen 4 (CTLA-4), programmed death 1 (PD-1), and their cognate ligands. Although systemic blocking of these molecules needs careful attention on the risk of uncontrolled immune activation, selective inhibition of negative signals in tumor-specific T cells by their genetic modification is an attractive approach to overcome immunological suppression in cancer patients. Here, we demonstrate the improved effector functions of tumor-specific CD4(+) and CD8(+) human T cells by small interfering RNA (siRNA) -mediated silencing of PD-1 ligands, PD-L1 or PD-L2. Tumor antigen MAGE-A4-specific human T-cell clones upregulated the expression of PD-1 ligands upon activation. siRNA-mediated knockdown of PD-L1 or -L2 enhanced the interferon-γ production and antigen-specific cytotoxicity of these cells. Peripheral blood mononuclear cells transduced with a retroviral vector encoding MAGE-A4-specific T-cell receptor α/β chains also increased their effector functions by this modification. These results suggest that siRNA-mediated knockdown of PD-1 ligands is an attractive strategy to inhibit a negative regulatory mechanism of tumor-specific T cells resulting in enhanced efficacy of adoptive T-cell therapy of cancer using genetically modified autologous lymphocytes.     

Enhancement of vaccine-induced primary and memory CD8(+) T-cell responses by soluble PD-1

Programmed death 1 (PD-1) signaling through its ligands, PD-L1 and PD-L2, has been known to negatively regulate T-cell responses. In addition, PD-L1 has been shown to interact with B7-1 costimulatory molecule to inhibit T-cell responses. Extensive studies have shown that PD-1/PD-L blockade restores exhausted T cells during chronic viral infections and tumors. In this study, we evaluated the effects of soluble PD-1 (sPD-1) as a blockade of PD-1 and PD-L1 on vaccine-elicited antigen-specific T-cell responses in mice. Coadministration of sPD-1 DNA with human papilloma virus-16 E7 DNA vaccine significantly enhanced E7-specific CD8(+) T-cell responses, resulting in potent antitumor effects against E7-expressing tumors. We also found that sPD-1, codelivered with adenovirus-based vaccine, could increase antigen-specific CD8(+) T-cell responses, indicating vaccine type-independent adjuvant effect of sPD-1. In addition, the frequency and functional activity of adoptively transferred OT-I cells, particularly memory CD8(+) T cells, were augmented by coadministration of sPD-1 DNA, which was closely associated with increased T-cell proliferation and reduced T-cell apoptosis through upregulation of Bcl-xL expression during T-cell activation. Codelivery of sPD-1 DNA also enhanced maturation of dendritic cells (DCs) in vivo which was accompanied by upregulation of DC maturation markers such as major histocompatibility complex class II. Taken together, our findings show that sPD-1 potently enhances codelivered antigen-specific CD8(+) T-cell responses and in vivo maturation of DCs during activation of naive CD8(+) T cells, suggesting that an immunization strategy with sPD-1 as an adjuvant can be used to increase antigen-specific T-cell immunity elicited by vaccination.     

Reversal of autoimmune diabetes by restoration of antigen-specific tolerance using genetically modified Lactococcus lactis in mice

Current interventions for arresting autoimmune diabetes have yet to strike the balance between sufficient efficacy, minimal side effects, and lack of generalized immunosuppression. Introduction of antigen via the gut represents an appealing method for induction of antigen-specific tolerance. Here, we developed a strategy for tolerance restoration using mucosal delivery in mice of biologically contained Lactococcus lactis genetically modified to secrete the whole proinsulin autoantigen along with the immunomodulatory cytokine IL-10. We show that combination therapy with low-dose systemic anti-CD3 stably reverted diabetes in NOD mice and increased frequencies of local Tregs, which not only accumulated in the pancreatic islets, but also suppressed immune response in an autoantigen-specific way. Cured mice remained responsive to disease-unrelated antigens, which argues against excessive immunosuppression. Application of this therapeutic tool achieved gut mucosal delivery of a diabetes-relevant autoantigen and a biologically active immunomodulatory cytokine, IL-10, and, when combined with a low dose of systemic anti-CD3, was well tolerated and induced autoantigen-specific long-term tolerance, allowing reversal of established autoimmune diabetes. Therefore, we believe this method could be an effective treatment strategy for type 1 diabetes in humans.     

In situ characterization of CD4+ T cell behavior in mucosal and systemic lymphoid tissues during the induction of oral priming and tolerance

The behavior of antigen-specific CD4+ T lymphocytes during initial exposure to antigen probably influences their decision to become primed or tolerized, but this has not been examined directly in vivo. We have therefore tracked such cells in real time, in situ during the induction of oral priming versus oral tolerance. There were marked contrasts with respect to rate and type of movement and clustering between naive T cells and those exposed to antigen in immunogenic or tolerogenic forms. However, the major difference when comparing tolerized and primed T cells was that the latter formed larger and longer-lived clusters within mucosal and peripheral lymph nodes. This is the first comparison of the behavior of antigen-specific CD4+ T cells in situ in mucosal and systemic lymphoid tissues during the induction of priming versus tolerance in a physiologically relevant model in vivo.     

Engagement of the PD-1 immunoinhibitory receptor by a novel B7 family member leads to negative regulation of lymphocyte activation

PD-1 is an immunoinhibitory receptor expressed by activated T cells, B cells, and myeloid cells. Mice deficient in PD-1 exhibit a breakdown of peripheral tolerance and demonstrate multiple autoimmune features. We report here that the ligand of PD-1 (PD-L1) is a member of the B7 gene family. Engagement of PD-1 by PD-L1 leads to the inhibition of T cell receptor-mediated lymphocyte proliferation and cytokine secretion. In addition, PD-1 signaling can inhibit at least suboptimal levels of CD28-mediated costimulation. PD-L1 is expressed by antigen-presenting cells, including human peripheral blood monocytes stimulated with interferon gamma, and activated human and murine dendritic cells. In addition, PD-L1 is expressed in nonlymphoid tissues such as heart and lung. The relative levels of inhibitory PD-L1 and costimulatory B7-1/B7-2 signals on antigen-presenting cells may determine the extent of T cell activation and consequently the threshold between tolerance and autoimmunity. PD-L1 expression on nonlymphoid tissues and its potential interaction with PD-1 may subsequently determine the extent of immune responses at sites of inflammation.     

Targeting the programmed cell death 1: programmed cell death ligand 1 pathway reverses T cell exhaustion in patients with sepsis

Introduction

A major pathophysiologic mechanism in sepsis is impaired host immunity which results in failure to eradicate invading pathogens and increased susceptibility to secondary infections. Although many immunosuppressive mechanisms exist, increased expression of the inhibitory receptor programmed cell death 1 (PD-1) and its ligand (PD-L1) are thought to play key roles. The newly recognized phenomenon of T cell exhaustion is mediated in part by PD-1 effects on T cells. This study tested the ability of anti-PD-1 and anti-PD-L1 antibodies to prevent apoptosis and improve lymphocyte function in septic patients.

Methods

Blood was obtained from 43 septic and 15 non-septic critically-ill patients. Effects of anti-PD-1, anti-PD-L1, or isotype-control antibody on lymphocyte apoptosis and interferon gamma (IFN-γ) and interleukin-2 (IL-2) production were quantitated by flow cytometry.

Results

Lymphocytes from septic patients produced decreased IFN-γ and IL-2 and had increased CD8 T cell expression of PD-1 and decreased PD-L1 expression compared to non-septic patients (P<0.05). Monocytes from septic patients had increased PD-L1 and decreased HLA-DR expression compared to non-septic patients (P<0.01). CD8 T cell expression of PD-1 increased over time in ICU as PD-L1, IFN-γ, and IL2 decreased. In addition, donors with the highest CD8 PD-1 expression together with the lowest CD8 PD-L1 expression also had lower levels of HLA-DR expression in monocytes, and an increased rate of secondary infections, suggestive of a more immune exhausted phenotype. Treatment of cells from septic patients with anti-PD-1 or anti-PD-L1 antibody decreased apoptosis and increased IFN-γ and IL-2 production in septic patients; (P<0.01). The percentage of CD4 T cells that were PD-1 positive correlated with the degree of cellular apoptosis (P<0.01).

Conclusions

In vitro blockade of the PD-1:PD-L1 pathway decreases apoptosis and improves immune cell function in septic patients. The current results together with multiple positive studies of anti-PD-1 and anti-PD-L1 in animal models of bacterial and fungal infections and the relative safety profile of anti-PD-1/anti-PD-L1 in human oncology trials to date strongly support the initiation of clinical trials testing these antibodies in sepsis, a disorder with a high mortality.     

Programmed cell death 1 forms negative costimulatory microclusters that directly inhibit T cell receptor signaling by recruiting phosphatase SHP2

Programmed cell death 1 (PD-1) is a negative costimulatory receptor critical for the suppression of T cell activation in vitro and in vivo. Single cell imaging elucidated a molecular mechanism of PD-1-mediated suppression. PD-1 becomes clustered with T cell receptors (TCRs) upon binding to its ligand PD-L1 and is transiently associated with the phosphatase SHP2 (Src homology 2 domain-containing tyrosine phosphatase 2). These negative costimulatory microclusters induce the dephosphorylation of the proximal TCR signaling molecules. This results in the suppression of T cell activation and blockade of the TCR-induced stop signal. In addition to PD-1 clustering, PD-1-TCR colocalization within microclusters is required for efficient PD-1-mediated suppression. This inhibitory mechanism also functions in PD-1(hi) T cells generated in vivo and can be overridden by a neutralizing anti-PD-L1 antibody. Therefore, PD-1 microcluster formation is important for regulation of T cell activation.     

B-Cell-Delivered Gene Therapy Induces Functional T Regulatory Cells and Leads to a Loss of Antigen-Specific Effector Cells

Previous reports have shown that B-cell-mediated gene therapy can induce tolerance in several animal models for autoimmune diseases and inhibitory antibody formation in hemophilia A mice. We know from our previous work that the induction of tolerance following B-cell therapy is dependent upon CD25⁺ regulatory T cells (Tregs). To extend these studies and identify the effects of this gene therapy protocol on the target CD4 T cells, we have adapted in vitro suppression assays using Tregs isolated from treated and control mice. Using carboxyfluorescein succinimidyl ester (CFSE) dilution as a measure of T-cell responsiveness to FVIII, we show that CD25⁺ Tregs from treated mice are more suppressive than those from control animals. To monitor the induction of antigen-specific Tregs, we repeated these studies in ovalbumin (OVA) peptide-specific DO11.10 T-cell receptor (TCR) transgenic mice. Tregs from DO11.10 mice treated with a tolerogenic OVA–Ig construct are better than polyclonal Tregs at suppressing the proliferation of responder cells stimulated with OVA peptide 323–339 (pOVA). Furthermore, we show that following B-cell therapy, there is an increase in antigen-specific FoxP3⁺ Tregs, and there is also a distinct decrease in antigen-specific CD4⁺ effector T cells. These changes in the lymphocyte population shift the balance away from effector function toward a tolerogenic phenotype.     

B7 but not intercellular adhesion molecule-1 costimulation prevents the induction of human alloantigen-specific tolerance

Presentation of antigen by the major histocompatibility complex to T lymphocytes without the requisite costimulatory signals does not induce an immune response but rather results in a state of antigen-specific unresponsiveness, termed anergy. To determine which costimulatory signals are critical for the T cell commitment to activation or anergy, we developed an in vitro model system that isolated the contributions of alloantigen and each candidate costimulatory molecule. Here, we show that transfectants expressing HLA-DR7 and either B7 or intercellular adhesion molecule 1 (ICAM-1) deliver independent costimulatory signals resulting in alloantigen-induced proliferation of CD4-positive T lymphocytes. Although equivalent in their ability to costimulate maximal proliferation of alloreactive T cells, B7 but not ICAM-1 induced detectable interleukin 2 secretion and prevented the induction of alloantigen-specific anergy. These results are consistent with the hypothesis that blockade of the ICAM-1:lymphocyte function-associated 1 pathway results in immunosuppression, whereas blockade of the B7:CD28/CTLA4 pathway results in alloantigen-specific anergy. This approach, using this model system, should facilitate the identification of critical costimulatory pathways which must be inhibited in order to induce alloantigen-specific tolerance before human organ transplantation.     

Molecular modeling and functional mapping of B7-H1 and B7-DC uncouple costimulatory function from PD-1 interaction

B7-H1 and B7-DC are ligands for PD-1, a receptor implicated in negative regulation of T and B cell functions. These ligands, however, also costimulate T cell responses. It remains elusive whether or not costimulation is mediated through PD-1. By comparative molecular modeling and site-directed mutagenesis, we found that nonconserved residues between these ligands on the A'GFCC'C" face mediate interaction with PD-1. This indicates significant structural heterogeneity of the interactions between PD-1 and its ligands. Importantly, ligand mutants with abolished PD-1 binding capacity could still costimulate proliferation and cytokine production of T cells from normal and PD-1-deficient mice. Our results reveal unique binding characteristics of B7-H1 and B7-DC and provide direct evidence for an independent costimulatory receptor other than PD-1.     

Virus-induced hepatocellular carcinomas cause antigen-specific local tolerance

T cell surveillance is often effective against virus-associated tumors because of their high immunogenicity. It is not clear why surveillance occasionally fails, particularly against hepatitis B virus– or hepatitis C virus–associated hepatocellular carcinoma (HCC). We established a transgenic murine model of virus-induced HCC by hepatocyte-specific adenovirus-induced activation of the oncogenic SV40 large T antigen (TAg). Adenovirus infection induced cytotoxic T lymphocytes (CTLs) targeted against the virus and TAg, leading to clearance of the infected cells. Despite the presence of functional, antigen-specific T cells, a few virus-infected cells escaped immune clearance and progressed to HCC. These cells expressed TAg at levels similar to HCC isolated from neonatal TAg-tolerant mice, suggesting that CTL clearance does not select for cells with low immunogenicity. Virus-infected mice revealed significantly greater T cell infiltration in early-stage HCC compared with that in late-stage HCC, demonstrating progressive local immune suppression through inefficient T cell infiltration. Programmed cell death protein-1 (PD-1) and its ligand PD-L1 were expressed in all TAg-specific CD8⁺ T cells and HCC, respectively, which contributed to local tumor-antigen-specific tolerance. Thus, we have developed a model of virus-induced HCC that may allow for a better understanding of human HCC.     

DNA vaccination with an insulin construct and a chimeric protein binding to both CTLA4 and CD40 ameliorates type 1 diabetes in NOD mice

Type 1 diabetes (T1D), a T-cell-mediated autoimmune disease, could be attributed to many defects in nonobese diabetic (NOD) mice, including deficient expressions of costimulatory molecules that impair antigen presentation. Thus, this deficient antigen presentation may result in a reduced ability to induce a tolerogenic response through negative selection/regulation of autoreactive T cells. Improperly activated T cells seem to be able to induce autoimmune responses causing diabetes. To re-establish tolerance to autoantigens by modulating costimulation, we constructed and tested a new type of DNA vaccine encoding a membrane-bound preproinsulin (mbPPI) and a chimeric gene vector encoding mutant B7.1/CD40L (mB7.1/CD40L) fusion protein. This mutant B7.1 binds CTLA4 but not CD28. We report that young NOD mice immunized with mbPPI along with mB7.1/CD40L DNA vectors significantly reduced diabetes incidence while treatment with CTLA4/IgG1 exacerbated diabetes. In conclusion, the combination of mbPPI and mB7.1/CD40L was able to protect against autoimmunity and diabetes in NOD mice possibly by promoting a more efficient presentation of autoantigen PPI and inducing specific tolerance to PPI by negatively regulating autoreactive T cells.