Enhancing Virus-Specific Immunity In Vivo by Combining Therapeutic Vaccination and PD-L1 Blockade in Chronic Hepadnaviral Infection

Hepatitis B virus (HBV) persistence is facilitated by exhaustion of CD8 T cells that express the inhibitory receptor programmed cell death-1 (PD-1). Improvement of the HBV-specific T cell function has been obtained in vitro by inhibiting the PD-1/PD-ligand 1 (PD-L1) interaction. In this study, we examined whether in vivo blockade of the PD-1 pathway enhances virus-specific T cell immunity and leads to the resolution of chronic hepadnaviral infection in the woodchuck model. The woodchuck PD-1 was first cloned, characterized, and its expression patterns on T cells from woodchucks with acute or chronic woodchuck hepatitis virus (WHV) infection were investigated. Woodchucks chronically infected with WHV received a combination therapy with nucleoside analogue entecavir (ETV), therapeutic DNA vaccination and woodchuck PD-L1 antibody treatment. The gain of T cell function and the suppression of WHV replication by this therapy were evaluated. We could show that PD-1 expression on CD8 T cells was correlated with WHV viral loads during WHV infection. ETV treatment significantly decreased PD-1 expression on CD8 T cells in chronic carriers. In vivo blockade of PD-1/PD-L1 pathway on CD8 T cells, in combination with ETV treatment and DNA vaccination, potently enhanced the function of virus-specific T cells. Moreover, the combination therapy potently suppressed WHV replication, leading to sustained immunological control of viral infection, anti-WHs antibody development and complete viral clearance in some woodchucks. Our results provide a new approach to improve T cell function in chronic hepatitis B infection, which may be used to design new immunotherapeutic strategies in patients.     

Characterization of T-cell response to woodchuck hepatitis virus core protein and protection of woodchucks from infection by immunization with peptides containing a T-cell epitope.

Specific activation of T cells appears to be a prerequisite for viral clearance during hepatitis B virus (HBV) infection. The T-cell response to HBV core protein is essential in determining an acute or chronic outcome of HBV infection, but how this immune response contributes to the course of infection remains unclear. This is due to results obtained from humans, which are restricted to phenomenological observations occurring during the clinical onset after HBV infection. Thus, a useful animal model is needed. Characterization of the T-cell response to the core protein (WHcAg) of woodchuck hepatitis virus (WHV) in woodchucks contributes to the understanding of these mechanisms. Therefore, we investigated the response of woodchuck peripheral blood mononuclear cells (PBMCs) to WHcAg and WHcAg-derived peptides, using our 5-bromo-2'-deoxyuridine assay. We demonstrated WHcAg-specific proliferation of PBMCs and nylon wool-nonadherent cells from acutely WHV-infected woodchucks. Using a cross-reacting anti-human T-cell (CD3) antiserum, we identified nonadherent cells as woodchuck T cells. T-cell epitope mapping with overlapping peptides, covering the entire WHcAg, revealed T-cell responses of acutely WHV-infected woodchucks to peptide1-20, peptide100-119, and peptide112-131. Detailed epitope analysis in the WHcAg region from amino acids 97 to 140 showed that T cells especially recognized peptide97-110. Establishment of polyclonal T-cell lines with WHcAg or peptide97-110 revealed reciprocal stimulation by peptide97-110 or WHcAg, respectively. We vaccinated woodchucks with peptide97-110 or WHcAg to prove the importance of this immunodominant T-cell epitope. All woodchucks immunized with peptide97-110 or WHcAg were protected. Our results show that the cellular immune response to WHcAg or to one T-cell epitope protects woodchucks from WHV infection.     

Upregulation of circulating PD-L1/PD-1 is associated with poor post-cryoablation prognosis in patients with HBV-related hepatocellular carcinoma

Background

The programmed cell death-1 receptor/programmed cell death-1 ligand (PD-1/PD-L1) pathway plays a crucial role in tumor evasion from host immunity. This study was designed to evaluate the association between circulating PD-L1/PD-1 and prognosis after cryoablation in patients with HBV-related hepatocellular carcinoma (HCC).

Methodology/Principal Findings

In the present study, 141 HBV-related HCC patients were enrolled and of those 109 patients received cryoablation. Circulating PD-L1/PD-1 expression was tested by flow cytometry, and 23 patients were simultaneously evaluated for intratumoral PD-L1 expression by immunohistochemical staining. Circulating PD-1/PD-L1 expression was associated with severity of diseases in patients with HCC, and the circulating PD-L1 expression was closely correlated with intratumoral PD-L1 expression. Of the clinical parameters, PD-1/PD-L1 expression was associated with tumor size, blood vessel invasion and BCLC staging. Moreover, PD-1/PD-L1 expression dropped after cryoablation while being elevated at the time of tumor recurrence. Patients with higher expression of circulating PD-L1, as well as circulating PD-1, had a significantly shorter overall survival and tumor-free survival than those with lower expression. Multivariate analysis confirmed that circulating PD-L1 could serve as an independent predictor of overall survival and tumor-recurrence survival in HCC patients after cryoablation.

Conclusions/Significance

Upregulation of circulating PD-L1/PD-1 is associated with poor post-cryoablation prognosis in patients with HBV-related hepatocellular carcinoma.     

Synovial T cell hyporesponsiveness to myeloid dendritic cells is reversed by preventing PD-1/PD-L1 interactions

Introduction

The aim of this study was to investigate PD-1/PD-L1 involvement in the hyporesponsiveness of rheumatoid arthritis (RA) synovial fluid (SF) CD4 T cells upon stimulation by thymic stromal lymphopoietin (TSLP)–primed CD1c myeloid dendritic cells (mDCs).

Methods

Expression of PD-1 on naïve (Tn), central memory (Tcm) and effector memory (Tem) CD4 T cell subsets was assessed by flow cytometry. PD-L1 expression and its regulation upon TSLP stimulation of mDCs from peripheral blood (PB) and SF of RA patients were investigated by quantitative RT-PCR and flow cytometry. The involvement of PD-1/PD-L1 interactions in SF T cell hyporesponsiveness upon (TSLP-primed) mDC activation was determined by cell culture in the presence of PD-1 blocking antibodies, with or without interleukin 7 (IL-7) as a recognized suppressor of PD-1 expression.

Results

PD-1 expression was increased on CD4 T cells derived from SF compared with PB of RA patients. TSLP increased PD-L1 mRNA expression in both PB and SF mDCs. PD-L1 protein expression was increased on SF mDCs compared with PB mDCs and was associated with T cell hyporesponsiveness. Blockade of PD-1, as well as IL-7 stimulation, during cocultures of memory T cells and (TSLP-primed) mDCs from RA patients significantly recovered T cell proliferation.

Conclusion

SF T cell hyporesponsiveness upon (TSLP-primed) mDC stimulation in RA joints is partially dependent on PD-1/PD-L1 interactions, as PD-1 and PD-L1 are both highly expressed on SF T cells and mDCs, respectively, and inhibiting PD-1 availability restores T cell proliferation. The potential of IL-7 to robustly reverse this hyporesponsiveness suggests that such proinflammatory cytokines in RA joints strongly contribute to memory T cell activation.     

Replication of naturally occurring woodchuck hepatitis virus deletion mutants in primary hepatocyte cultures and after transmission to naive woodchucks

Woodchuck hepatitis virus (WHV) mutants with core internal deletions (CID) occur naturally in chronically WHV-infected woodchucks, as do hepatitis B virus mutants in humans. We studied the replication of WHV deletion mutants in primary woodchuck hepatocyte cultures and in vivo after transmission to naive woodchucks. By screening 14 wild-caught, chronically WHV-infected woodchucks, two woodchucks, WH69 and WH70, were found to harbor WHV CID mutants. Consistent with previous results, WHV CID mutants from both animals had deletions of variable lengths (90 to 135 bp) within the middle of the WHV core gene. In woodchuck WH69, WHV CID mutants represented a predominant fraction of the viral population in sera, normal liver tissues, and to a lesser extent, in liver tumor tissues. In primary hepatocytes of WH69, the replication of wild-type WHV and CID mutants was maintained at least for 7 days. Although WHV CID mutants were predominant in fractions of cellular WHV replicative intermediates, mutant covalently closed circular DNAs (cccDNAs) appeared to be a small part of cccDNA-enriched fractions. Analysis of cccDNA-enriched fractions from liver tissues of other woodchucks confirmed that mutant cccDNA represents only a small fraction of the total cccDNA pool. Four naive woodchucks were inoculated with sera from woodchuck WH69 or WH70 containing WHV CID mutants. All four woodchucks developed viremia after 3 to 4 weeks postinoculation (p.i.). They developed anti-WHV core antigen (WHcAg) antibody, lymphoproliferative response to WHcAg, and anti-WHV surface antigen. Only wild-type WHV, but no CID mutant, was found in sera from these woodchucks. The WHV CID mutant was also not identified in liver tissue from one woodchuck sacrificed in week 7 p.i. Three remaining woodchucks cleared WHV. Thus, the presence of WHV CID mutants in the inocula did not significantly change the course of acute self-limiting WHV infection. Our results indicate that the replication of WHV CID mutants might require some specific selective conditions. Further investigations on WHV CID mutants will allow us to have more insight into hepadnavirus replication.     

Molecular characterization of woodchuck type I interferons and their expression by woodchuck peripheral blood lymphocytes

Interferon (IFN)-alpha and -beta are important antiviral mediators. IFN-alpha is widely used for the treatment of chronic hepatitis B. In our previous studies, a subtype of woodchuck IFN-alpha (wIFN-alpha) was characterized and has been shown to be active in suppressing the replication of woodchuck hepatitis virus (WHV) in vitro and vivo. Here, we refined the analysis of the IFN-alpha/beta system of the woodchuck and studied the expression of wIFN-alpha/beta in peripheral blood lymphocytes (PBLs) from na?ve and WHV-infected woodchucks. A number of wIFN-alpha genes were sequenced and could be classified into 10 subtypes and 3 pseudotypes. The biological activity of different subtypes of wIFN-alpha was demonstrated by their ability to protect woodchuck cells against encephalomyocarditis virus infection and to induce MxA expression in woodchuck cells. Additionally, a partial sequence of wIFN-beta was characterized. A subtyping method for wIFN-alpha based on restriction length polymorphism analysis was developed. Further, the expression of wIFN by woodchuck PBLs after stimulation with polyI/C was investigated. The maximal production of wIFN by woodchuck PBLs occurred within the first 48 h after addition poly I/C. The wIFN-alpha subtypes 1, 4, and 5 were found to be produced by poly I/C-stimulated woodchuck PBLs, indicating a selective expression of wIFN-alpha subtypes. PBLs from chronically WHV-infected woodchucks showed a reduced ability to produce wIFN when stimulated with poly I/C. The results suggest that woodchucks with chronic WHV infection have impaired immunological responses to poly I/C.     

PD-1, PD-L1 and PD-L2 Gene Expression on T-Cells and Natural Killer Cells Declines in Conjunction with a Reduction in PD-1 Protein during the Intensive Phase of Tuberculosis Treatment

Background

The PD-1 axis is a cell intrinsic immunoregulatory pathway that mediates T cell exhaustion in chronic infection particularly in some viral infections. We hypothesized that PD-1, PD-L1 and PD-L2 would be highly expressed in untreated tuberculosis patients compared to controls due to their chronic infection and would decrease with successful TB treatment.

Materials and Methods

Untreated tuberculosis patients (n = 26) were recruited at diagnosis and followed up during treatment. Household contacts (n = 24) were recruited to establish baseline differences. Blood gene expression ex vivo was investigated using qRT-PCR. Flow cytometry was performed to establish protein expression patterns.

Results

PD-L1 gene expression was found to be elevated in active TB disease; however, this was not observed for PD-1 or PD-L2. The intensive phase of TB treatment was associated with a significant decline in PD-1, PD-L1 and PD-L2 gene expression. PD-1 protein expression on the surface of NK cells, CD8⁺ and CD4⁺ T cells was similar in patients with active TB disease compared to controls but declined with successful TB treatment, with the greatest decline occurring on the NK cells followed by CD8⁺ T cells and then CD4⁺ T cells. Granzyme B/PD-1 co-expression declined with successful intensive phase treatment.

Conclusion

Modulation of PD-1/PD-L1 pathway through TB treatment indicates changes in the peripheral T cell response caused by live Mycobacterium tuberculosis (Mtb) followed by the response to dead bacilli, antigen-release and immuno-pathology resolution. The PD-1 axis could be a host drug target for immunomodulatory treatments in the future.     

T cells from Programmed Death-1 deficient mice respond poorly to Mycobacterium tuberculosis infection

Background

Programmed Death-1 (PD-1; CD279) receptor molecule is widely believed to be a negative regulator predominantly expressed by exhausted/activated mouse T cells. Upon interaction with its ligands, PD-L1 and PD-L2, PD-1 inhibits activation of T cells and cytokine production, which has been documented in various viral and fungal infections as well as in vitro studies. Therefore, inhibition of T cell responses by PD-1 resulted in disease resistance in a variety of mouse infection models studied heretofore.

Methodology/Principal Findings

Here, we report that PD-1 deficient (PD-1^−/−) mice infected with Mycobacterium tuberculosis (M. tb) H37Rv by the aerosol route have increased susceptibility as compared with their wild type littermates. Surprisingly, M. tb antigen-specific T cell proliferation was dramatically reduced in PD-1 deficient animals compared with wild-type littermates, and this was due to increased numbers of regulatory T cells (Tregs) and recruitment of mesenchymal stem cells. Furthermore, PD-1^−/− mice exhibited decreases in the autophagy-induced LC3-B marker protein in macrophages.

Conclusions/Significance

Our findings suggest that PD-1 does not play an inhibitory role during M. tb infection and instead promotes mycobacterial clearance in mice.     

Immunization of Woodchucks with Plasmids Expressing Woodchuck Hepatitis Virus (WHV) Core Antigen and Surface Antigen Suppresses WHV Infection

DNA vaccination can induce humoral and cellular immune response to viral antigens and confer protection to virus infection. In woodchucks, we tested the protective efficacy of immune response to woodchuck hepatitis core antigen (WHcAg) and surface antigen (WHsAg) of woodchuck hepatitis virus (WHV) elicited by DNA-based vaccination. Plasmids pWHcIm and pWHsIm containing WHV c- or pre-s2/s genes expressed WHcAg and WHsAg in transient transfection assays. Pilot experiments in mice revealed that a single intramuscular injection of 100 μg of plasmid pWHcIm DNA induced an anti-WHcAg titer over 1:300 that was enhanced by boost injections. However, two injections of 100 μg of pWHcIm did not induce detectable anti-WHcAg in woodchucks. With an increase in the dose to 1 mg of pWHcIm per injection, transient anti-WHcAg response and WHcAg-specific proliferation of peripheral mononuclear blood cells (PMBCs) appeared in woodchucks after repeated immunizations. Four woodchucks vaccinated with pWHcIm were challenged with 10⁴ or 10⁵ of the WHV 50% infective dose. They remained negative for markers of WHV replication (WHV DNA and WHsAg) in peripheral blood and developed anti-WHs in week 5 after challenge. In contrast, woodchucks not immunized or immunized with the control vector pcDNA3 developed acute WHV infection. Two woodchucks immunized with 1 mg of pWHsIm developed WHsAg-specific proliferative response of PBMCs but no measurable anti-WHsAg response. A rapid anti-WHsAg response developed during week 2 after virus challenge. Neither woodchuck developed any signs of WHV infection. These data indicate that DNA-based vaccination with WHcAg and WHsAg can elicit immunity to WHV infection.     

Inhibitory Phenotype of HBV-Specific CD4+ T-Cells Is Characterized by High PD-1 Expression but Absent Coregulation of Multiple Inhibitory Molecules

Background

T-cell exhaustion seems to play a critical role in CD8⁺ T-cell dysfunction during chronic viral infections. However, up to now little is known about the mechanisms underlying CD4⁺ T-cell dysfunction during chronic hepatitis B virus (CHB) infection and the role of inhibitory molecules such as programmed death 1 (PD-1) for CD4⁺ T-cell failure.

Methods

The expression of multiple inhibitory molecules such as PD-1, CTLA-4, TIM-3, CD244, KLRG1 and markers defining the grade of T-cell differentiation as CCR7, CD45RA, CD57 and CD127 were analyzed on virus-specific CD4⁺ T-cells from peripheral blood using a newly established DRB1*01-restricted MHC class II Tetramer. Effects of in vitro PD-L1/2 blockade were defined by investigating changes in CD4⁺ T-cell proliferation and cytokine production.

Results

CD4⁺ T-cell responses during chronic HBV infection was characterized by reduced Tetramer⁺CD4⁺ T-cell frequencies, effector memory phenotype, sustained PD-1 but low levels of CTLA-4, TIM-3, KLRG1 and CD244 expression. PD-1 blockade revealed individualized patterns of in vitro responsiveness with partly increased IFN-γ, IL-2 and TNF-α secretion as well as enhanced CD4⁺ T-cell expansion almost in treated patients with viral control.

Conclusion

HBV-specific CD4⁺ T-cells are reliably detectable during different courses of HBV infection by MHC class II Tetramer technology. CD4⁺ T-cell dysfunction during chronic HBV is basically linked to strong PD-1 upregulation but absent coregulation of multiple inhibitory receptors. PD-L1/2 neutralization partly leads to enhanced CD4⁺ T-cell functionality with heterogeneous patterns of CD4⁺ T-cell rejunivation.     

Primary human hepatocytes are susceptible to infection by hepatitis delta virus assembled with envelope proteins of woodchuck hepatitis virus

Hepatitis B virus (HBV) and hepatitis delta virus (HDV) share the HBV envelope proteins. When woodchucks chronically infected with woodchuck hepatitis virus (WHV) are superinfected with HDV, they produce HDV with a WHV envelope, wHDV. Several lines of evidence are provided that wHDV infects not only cultured primary woodchuck hepatocytes (PWH) but also primary human hepatocytes (PHH). Surprisingly, HBV-enveloped HDV (hHDV) and wHDV infected PHH with comparable efficiencies; however, hHDV did not infect PWH. The basis for these host range specificities was investigated using as inhibitors peptides bearing species-specific pre-S (where S is the small envelope protein) sequences. It was found that pre-S1 contributed to the ability of wHDV to infect both PHH and PWH. In addition, the inability of hHDV to infect PWH was not overcome using a chimeric form of hHDV containing WHV S protein, again supporting the essential role of pre-S1 in infection of target cells. One interpretation of these data is that host range specificity of HDV is determined entirely by pre-S1 and that the WHV and HBV pre-S1 proteins recognize different receptors on PHH.     

PD-1 and PD-L1 Expression in NSCLC Indicate a Favorable Prognosis in Defined Subgroups

Background

Immunotherapy can become a crucial therapeutic option to improve prognosis for lung cancer patients. First clinical trials with therapies targeting the programmed cell death receptor PD-1 and its ligand PD-L1 have shown promising results in several solid tumors. However, in lung cancer the diagnostic, prognostic and predictive value of these immunologic factors remains unclear.

Method

The impact of both factors was evaluated in a study collective of 321 clinically well-annotated patients with non-small lung cancer (NSCLC) using immunohistochemistry.

Results

PD-1 expression by tumor infiltrating lymphocytes (TILs) was found in 22%, whereas tumor cell associated PD-L1 expression was observed in 24% of the NSCLC tumors. In Fisher’s exact test a positive correlation was found for PD-L1 and Bcl-xl protein expression (p = 0.013). Interestingly, PD-L1 expression on tumor cells was associated with improved overall survival in pulmonary squamous cell carcinomas (SCC, p = 0.042, log rank test), with adjuvant therapy (p = 0.017), with increased tumor size (pT2-4, p = 0.039) and with positive lymph node status (pN1-3, p = 0.010). These observations were confirmed by multivariate cox regression models.

Conclusion

One major finding of our study is the identification of a prognostic implication of PD-L1 in subsets of NSCLC patients with pulmonary SCC, with increased tumor size, with a positive lymph node status and NSCLC patients who received adjuvant therapies. This study provides first data for immune-context related risk stratification of NSCLC patients. Further studies are necessary both to confirm this observation and to evaluate the predictive value of PD-1 and PD-L1 in NSCLC in the context of PD-1 inhibition.     

Upregulation of Programmed Death-1 and Its Ligand in Cardiac Injury Models: Interaction with GADD153

Purpose

Programmed Death-1 (PD-1) and its ligand, PD-L1, are regulators of immune/ inflammatory mechanisms. We explored the potential involvement of PD-1/PD-L1 pathway in the inflammatory response and tissue damage in cardiac injury models.

Experimental Design

Ischemic-reperfused and cryoinjured hearts were processed for flow cytometry and immunohistochemical studies for determination of cardiac PD-1 and PD-L1 in the context of assessment of the growth arrest- and DNA damage-inducible protein 153 (GADD153) which regulates both inflammation and cell death. Further, we explored the potential ability of injured cardiac cells to influence proliferation of T lymphocytes.

Results

The isolated ischemic-reperfused hearts displayed marked increases in expression of PD-1 and PD-L1 in cardiomyocytes; however, immunofluorescent studies indicate that PD-1 and PD-L1 are not primarily co-expressed on the same cardiomyocytes. Upregulation of PD-1/PD-L1 was associated with a) marked increases in GADD153 and interleukin (IL)-17 but a mild increase in IL-10 and b) disruption of mitochondrial membrane potential (ψ_m) as well as apoptotic and necrotic cell death. Importantly, while isotype matching treatment did not affect the aforementioned changes, treatment with the PD-L1 blocking antibody reversed those effects in association with marked cardioprotection. Further, ischemic-reperfused cardiac cells reduced proliferation of T lymphocytes, an effect partially reversed by PD-L1 antibody. Subsequent studies using the cryoinjury model of myocardial infarction revealed significant increases in PD-1, PD-L1, GADD153 and IL-17 positive cells in association with significant apoptosis/necrosis.

Conclusions

The data suggest that upregulation of PD-1/PD-L1 pathway in cardiac injury models mediates tissue damage likely through a paracrine mechanism. Importantly, inhibition of T cell proliferation by ischemic-reperfused cardiac cells is consistent with the negative immunoregulatory role of PD-1/PD-L1 pathway, likely reflecting an endogenous cardiac mechanism to curtail the deleterious impact of infiltrating immune cells to the damaged myocardium. The balance of these countervailing effects determines the extent of cardiac injury.     

Lack of an association of PD-1 and its ligand genes with Behcet's disease in a Chinese Han population

Background

Behcet''s disease is a chronic, multi-systemic autoimmune disease. Programmed cell death 1 (PD-1) gene is one of non-human leucocyte antigen genes. It has been demonstrated to be associated with several autoimmune diseases. However, only a few studies have addressed the association of ligand genes of PD-1, PD-L1 and PD-L2 with autoimmune disease. The purpose of this study was to analyze the potential association of the PD-1 and its ligand genes with Behcet''s disease in a Chinese Han population.

Methodology/Principal Findings

Four single-nucleotide polymorphism (SNPs) rs2227981 and rs10204525 of PD-1, rs1970000 of PD-L1 and rs7854303 of PD-L2 were genotyped in 405 Behcet''s patients and 414 age-, sex-, ethnic-matched healthy controls using polymerase chain reaction-restriction fragment length polymorphism assay. The results revealed that there were no significant differences in the genotype and allele frequencies of PD-1 rs2227981 and rs10204525 between the Behcet''s patients and controls. A similar result was found for PD-L1 rs1970000 versus healthy controls. Only the C allele and the CC genotype of PD-L2 rs7854303 were identified in patients and controls. Stratification analysis based on gender and clinical findings did not show any associations between PD-1 or its ligand polymorphisms and Behcet''s disease.

Conclusions/Significance

None of the currently studied SNPs, PD-1 rs2227981 and rs10204525, PD-L1 rs1970000 and PD-L2 rs7854303, are associated with the susceptibility to Behcet''s disease in a Chinese Han population. More studies are needed to confirm these findings in Behcet''s patients with other ethnic backgrounds.     

PD-L1 and PD-1 expression are correlated with distinctive clinicopathological features in papillary thyroid carcinoma

Background

Immune checkpoint blockade targeting PD-1/PD-L1 has shown efficacy in several types of cancers. However, the correlation between PD-L1/PD-1 expression and the specific clinicopathological features in papillary thyroid carcinoma (PTC) has not been investigated.

Methods

We examined the immunohistochemical expression of PD-L1, PD-1, and BRAF V600E on whole-tissue sections from 126 cases of primary PTC more than 1 cm in size. The correlation between the PD-L1/PD-1 expression and the clinicopathological features was evaluated.

Results

PD-L1 was positively expressed in 53.2% PTCs, and its expression was positively correlated with rich tumor-infiltrating lymphocytes (TILs), background chronic lymphocytic thyroiditis (CLT), female gender, absence of psammoma bodies, and PD-1 expression. Among these parameters, rich TILs, female gender, and absence of psammoma bodies were independent factors affecting PD-L1 expression on the multivariate logistic regression analysis. PD-1 expression was detected in the TILs and was positively correlated with rich TILs, background CLT, and absence of stromal calcification. Lack of stromal calcification was an independent factor affecting PD-1 expression. Neither PD-L1 nor PD-1 expression showed significant correlation with BRAF V600E expression.

Conclusions

Our results show that the distinctive pathological features of PTCs, including TILs, background CLT, female gender, psammoma bodies, and stromal calcification, are useful parameters for predicting PD-L1 or PD-1 expression.

     

Type III Interferons,IL-28 and IL-29, Are Increased in Chronic HCV Infection and Induce Myeloid Dendritic Cell-Mediated FoxP3+ Regulatory T Cells

Background & Aims

Hepatitis C virus (HCV) is difficult to eradicate and type III interferons (IFN-λ, composed of IL-28A, IL-28B and IL-29) are novel therapeutic candidates. We hypothesized that IFN-λ have immunomodulatory effects in HCV- infected individuals.

Materials and Methods

We analyzed the expression of IFN-λ and its receptor (composed of IL-10R2 and IFN-λR subunits) in the blood and livers of patients with chronic (c)HCV infection compared to controls (those who cleared HCV by sustained virological response, SVR, and those with liver inflammation of non-viral origin, non-alcoholic steatohepatitis, NASH). We also compared the proliferative capacity of dendritic cells (DCs) obtained from healthy individuals and those with chronic HCV using a mixed leukocyte reaction combined with 3H-Td incorporation. In addition, the composition of the IFN-λ receptor (IFN-λR) on myeloid DCs, plasmacytoid DCs, PBMCs, and T cells was determined by FACS analysis.

Results

We report that the expression of IFN-λ protein in serum and mRNA in liver is increased in cHCV patients, but not in those with HCV SVR or NASH, compared to controls. Liver level of IFN-λR mirrored the expression of serum IFN-λ and was higher in cHCV, compared to controls and HCV-SVR patients, suggesting that elevation of IFN-λ and IFN-λR are HCV-dependent. We further identified that innate immune cell populations expressed complete IFN-λ receptor. In vitro, recombinant IFN-λ promoted differentiation of monocyte-derived dendritic cells (DCs) into a phenotype with low T cell stimulatory capacity and high PD-L1 expression, which further promoted expansion of existing regulatory T cells. IFN-λ-DCs failed to induce de novo generation of regulatory T cells. The inhibitory capacity of IFN-λ-DCs was counteracted by recombinant IL-12 and by neutralization of the PD-1/PD-L1 system.

Conclusions

Our novel findings of the immunomodulatory effect of IFN-λ contribute to the understanding of the anti-inflammatory and/or anti-viral potential of IFN-λ in cHCV.     

Relationship of Glucocorticoid Receptor Expression in Peripheral Blood Mononuclear Cells and the Cochlea of Guinea Pigs and Effects of Dexamethasone Administration

Background

Glucocorticoids (GCs) are widely used to treat sudden sensorineural hearing loss (SSNHL) and significantly improve hearing. However, GC insensitivity has been observed in some patients of SSNHL.

Objective

To study the correlation between GR expression in peripheral blood mononuclear cells (PBMCs) and in the cochlea of guinea pigs at mRNA and protein levels.

Methods

One group of guinea pigs received dexamethasone (10 mg/kg/day) intraperitoneally for 7 consecutive days (dexamethasone group), and another group of guinea pigs received normal saline (control group). Real time PCR and Western blotting were used to detect the expression of GR mRNA and GR protein in PBMCs and the cochleae.

Results

The GR mRNA and GR protein were detected in both PBMCs and the cochlear tissue of guinea pigs. GR mRNA and GR protein levels in PBMCs were positively correlated with those in the cochlea. The expression of GR mRNA and GR protein was significantly increased in the dexamethasone group compared to the control group.

Conclusions

Levels of GR mRNA and GR protein in the PBMCs were positively correlated with those in the cochlea of guinea pigs. Systemic dexamethasone treatment can significantly up-regulate GR expression in PBMCs and in the cochlea. Measurement of the GR level in PBMCs could be used as an indicator of GR level in the cochlea.     

T-Cell Response to Woodchuck Hepatitis Virus (WHV) Antigens during Acute Self-Limited WHV Infection and Convalescence and after Viral Challenge

The infection of woodchucks with woodchuck hepatitis virus (WHV) provides an experimental model to study early immune responses during hepadnavirus infection that cannot be tested in patients. The T-cell response of experimentally WHV-infected woodchucks to WHsAg, rWHcAg, and WHcAg peptides was monitored by observing 5-bromo-2′-deoxyuridine and [2-³H]adenine incorporation. The first T-cell responses were directed against WHsAg 3 weeks after infection; these were followed by responses to rWHcAg including the immunodominant T-cell epitope of WHcAg (amino acids 97 to 110). Maximal proliferative responses were detected when the animals seroconvered to anti-WHs and anti-WHc (week 6). A decrease in the T-cell response to viral antigens coincided with clearance of viral DNA. Polyclonal rWHcAg-specific T-cell lines were established 6, 12, 18, and 24 weeks postinfection, and their responses to WHcAg peptides were assessed. Five to seven peptides including the immunodominant epitope were recognized throughout the observation period (6 months). At 12 months after infection, T-cell responses to antigens and peptides were not detected. Reactivation of T-cell responses to viral antigens and peptides occurred within 7 days after challenge of animals with WHV. These results demonstrate that a fast and vigorous T-cell response to WHsAg, rWHcAg, and amino acids 97 to 110 of the WHcAg occurs within 3 weeks after WHV infection. The peak of this response was associated with viral clearance and may be crucial for recovery from infection. One year after infection, no proliferation of T cells in response to antigens was observed; however, the WHV-specific T-cell response was reactivated after challenge of woodchucks with WHV and may be responsible for protection against WHV reinfection.     

Woodchuck gamma interferon upregulates major histocompatibility complex class I transcription but is unable to deplete woodchuck hepatitis virus replication intermediates and RNAs in persistently infected woodchuck primary hepatocytes.

Gamma interferon (IFN-gamma) is an important mediator with multiple functions in the host defense against viral infection. IFN-gamma, in concert with tumor necrosis factor alpha (TNF-alpha), leads to a remarkable reduction of intrahepatic replication intermediates and specific mRNAs of hepatitis B virus (HBV) by a noncytolytic mechanism in the transgenic mouse model. Thus, it is rational to evaluate the potential value of IFN-gamma for the treatment of chronic HBV infection. In the present study, we expressed recombinant woodchuck IFN-gamma (wIFN-gamma) in Escherichia coli and mammalian cells. wIFN-gamma protected woodchuck cells against infection of murine encephalomyocarditis virus in a species-specific manner. It upregulated the mRNA level of the woodchuck major histocompatibility complex class I (MHC-I) heavy chain in permanent woodchuck WH12/6 cells and regulated differentially the gene expression. However, the level of the replication intermediates and specific RNAs of woodchuck hepatitis virus (WHV) in persistently WHV-infected primary woodchuck hepatocytes did not change despite a treatment with 1,000 U of wIFN-gamma per ml or with a combination of wIFN-gamma and woodchuck TNF-alpha. Rather, hepatocytes derived from chronic carriers had an elevated level of the MHC-I heavy-chain mRNAs, most probably due to the exposure to inflammatory cytokines in vivo. Treatment with high doses of wIFN-gamma led to an abnormal cell morphology and loss of hepatocytes. Thus, wIFN-gamma regulates the gene expression in woodchuck hepatocytes but could not deplete WHV replication intermediates and mRNAs in persistently infected hepatocytes. The cellular response to wIFN-gamma may be changed in hepatocytes from chronically WHV-infected woodchucks. It should be clarified in the future whether the continuous exposure of hepatocytes to inflammatory cytokines or the presence of viral proteins leads to changes of the cellular response to wIFN-gamma.