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Shahriar Behboudi - Top 30 Publications

Association of Marek's Disease induced immunosuppression with activation of a novel regulatory T cells in chickens.

Marek's Disease Virus (MDV) is an alphaherpesvirus that infects chickens, transforms CD4+ T cells and causes deadly lymphomas. In addition, MDV induces immunosuppression early during infection by inducing cell death of the infected lymphocytes, and potentially due to activation of regulatory T (Treg)-cells. Furthermore, immunosuppression also occurs during the transformation phase of the disease; however, it is still unknown how the disease can suppress immune response prior or after lymphoma formation. Here, we demonstrated that chicken TGF-beta+ Treg cells are found in different lymphoid tissues, with the highest levels found in the gut-associated lymphoid tissue (cecal tonsil: CT), fostering an immune-privileged microenvironment exerted by TGF-beta. Surprisingly, significantly higher frequencies of TGF-beta+ Treg cells are found in the spleens of MDV-susceptible chicken lines compared to the resistant line, suggesting an association between TGF-beta+ Treg cells and host susceptibility to lymphoma formation. Experimental infection with a virulent MDV elevated the levels of TGF-beta+ Treg cells in the lungs as early as 4 days post infection, and during the transformation phase of the disease in the spleens. In contrast to TGF-beta+ Treg cells, the levels of CD4+CD25+ T cells remained unchanged during the infection and transformation phase of the disease. Furthermore, our results demonstrate that the induction of TGF-beta+ Treg cells is associated with pathogenesis of the disease, as the vaccine strain of MDV did not induce TGF-beta+ Treg cells. Similar to human haematopoietic malignant cells, MDV-induced lymphoma cells expressed high levels of TGF-beta but very low levels of TGF-beta receptor I and II genes. The results confirm that COX-2/ PGE2 pathway is involved in immunosuppression induced by MDV-lymphoma cells. Taken together, our results revealed a novel TGF-beta+ Treg subset in chickens that is activated during MDV infection and tumour formation.

Ex Vivo PD-L1/PD-1 Pathway Blockade Reverses Dysfunction of Circulating CEA-Specific T Cells in Pancreatic Cancer Patients.

Purpose: Carcinoembryonic antigen (CEA) is a candidate target for cellular immunotherapy of pancreatic cancer. In this study, we have characterized the antigen-specific function of autologous cytotoxic T lymphocytes (CTL) specific for the HLA-A2-restricted peptide, pCEA691-699, isolated from the peripheral T-cell repertoire of pancreatic cancer patients and sought to determine if ex vivo PD-L1 and TIM-3 blockade could enhance CTL function.Experimental Design: CD8+ T-cell lines were generated from peripheral blood mononuclear cells of 18 HLA-A2+ patients with pancreatic cancer and from 15 healthy controls. In vitro peptide-specific responses were evaluated by flow cytometry after staining for intracellular cytokine production and carboxy fluorescein succinimydyl ester cytotoxicity assays using pancreatic cancer cell lines as targets.Results: Cytokine-secreting functional CEA691-specific CTL lines were successfully generated from 10 of 18 pancreatic cancer patients, with two CTL lines able to recognize and kill both CEA691 peptide-loaded T2 cells and CEA+ HLA-A2+ pancreatic cancer cell lines. In the presence of ex vivo PD-L1 blockade, functional CEA691-specific CD8+ T-cell responses, including IFNγ secretion and proliferation, were enhanced, and this effect was more pronounced on Ag-specific T cells isolated from tumor draining lymph nodes.Conclusions: These data demonstrate that CEA691-specific CTL can be readily expanded from the self-restricted T-cell repertoire of pancreatic cancer patients and that their function can be enhanced by PD-L1 blockade. Clin Cancer Res; 23(20); 6178-89. ©2017 AACR.

Marek's disease in chickens: a review with focus on immunology.

Marek's disease (MD), caused by Marek's disease virus (MDV), is a commercially important neoplastic disease of poultry which is only controlled by mass vaccination. Importantly, vaccines that can provide sterile immunity and inhibit virus transmission are lacking; such that vaccines are only capable of preventing neuropathy, oncogenic disease and immunosuppression, but are unable to prevent MDV transmission or infection, leading to emergence of increasingly virulent pathotypes. Hence, to address these issues, developing more efficacious vaccines that induce sterile immunity have become one of the important research goals for avian immunologists today. MDV shares very close genomic functional and structural characteristics to most mammalian herpes viruses such as herpes simplex virus (HSV). MD also provides an excellent T cell lymphoma model for gaining insights into other herpesvirus-induced oncogenesis in mammals and birds. For these reasons, we need to develop an in-depth knowledge and understanding of the host-viral interaction and host immunity against MD. Similarly, the underlying genetic variation within different chicken lines has a major impact on the outcome of infection. In this review article, we aim to investigate the pathogenesis of MDV infection, host immunity to MD and discuss areas of research that need to be further explored.

1α,25(OH)2 Vitamin D3 Modulates Avian T Lymphocyte Functions without Inducing CTL Unresponsiveness.

1,25-Dihydroxyvitamin D3 (Vitamin D) is a naturally synthesized fat soluble vitamin shown to have immunomodulatory, anti-inflammatory and cancer prevention properties in human and murine models. Here, we studied the effects of Vitamin D on the functional abilities of avian T lymphocytes using chicken Interferon (IFN)-γ ELISPOT assay, BrdU proliferation assay, Annexin V apoptosis assay and PhosFlow for detecting phosphorylated signalling molecules. The results demonstrate that Vitamin D significantly inhibited the abilities of T lymphocytes to produce IFN-γ and proliferate in vitro (P≤0.05), but retained their ability to undergo degranulation, which is a maker for cytotoxicity of these cells. Similarly, Vitamin D did not inhibit Extracellular signal-Regulated Kinase (ERK) 1/2 phosphorylation, a key mediator in T cell signalling, in the stimulated T lymphocytes population, while reduced ERK1/2 phosphorylation levels in the unstimulated cells. Our data provide evidence that Vitamin D has immuno-modulatory properties on chicken T lymphocytes without inducing unresponsiveness and by limiting immuno-pathology can promote protective immunity against infectious diseases of poultry.

Induction of antiviral responses against avian influenza virus in embryonated chicken eggs with toll-like receptor ligands.

Early responses against viruses, such as avian influenza virus (AIV), may be induced by Toll-like receptor (TLR) pathways. In the present study, an in ovo model was employed to study the antiviral activities of TLR ligands. It was hypothesized that administration of TLR ligands in ovo at the appropriate dose and time can reduce AIV titer in embryonated chicken eggs. Moreover, the study aimed to determine the mechanisms involved in the TLR-mediated antiviral responses in the chorioallantoic membrane (CAM). Embryonated eggs (10-14 day old) were treated with TLR2, 4, 7, and 21 ligands using different doses and times pre- and post-AIV infection. The results revealed that treatment of embryonated chicken eggs with TLR ligands reduced AIV replication. Further analysis showed that TLR ligands induced interferon (IFN)-γ and IFN stimulatory genes in the CAM, which may have played a role in the reduction of the AIV titer. The timing and dose of TLR ligands administration had significant impacts on the outcome of the treated eggs. In conclusion, the present study demonstrated that the in ovo route may be employed to determine the antiviral characteristics of TLR ligands against AIV.

TLR ligands induce antiviral responses in chicken macrophages.

Chicken macrophages express several receptors for recognition of pathogens, including Toll-like receptors (TLRs). TLRs bind to pathogen-associated molecular patterns (PAMPs) derived from bacterial or viral pathogens leading to the activation of macrophages. Macrophages play a critical role in immunity against viruses, including influenza viruses. The present study was designed to test the hypothesis that treatment of chicken macrophages with TLR ligands reduces avian influenza replication. Furthermore, we sought to study the expression of some of the key mediators involved in the TLR-mediated antiviral responses of macrophages. Chicken macrophages were treated with the TLR2, 3, 4, 7 and 21 ligands, Pam3CSK4, poly(I:C), LPS, R848 and CpG ODN, respectively, at different doses and time points pre- and post-H4N6 avian influenza virus (AIV) infection. The results revealed that pre-treatment of macrophages with Pam3CSK4, LPS and CpG ODN reduced the replication of AIV in chicken macrophages. In addition, the relative expression of genes involved in inflammatory and antiviral responses were quantified at 3, 8 and 18 hours post-treatment with the TLR2, 4 and 21 ligands. Pam3CSK4, LPS and CpG ODN increased the expression of interleukin (IL)-1β, interferon (IFN)-γ, IFN-β and interferon regulatory factor (IFR) 7. The expression of these genes correlated with the reduction of viral replication in macrophages. These results shed light on the process of immunity to AIV in chickens.

The effects of administration of ligands for Toll-like receptor 4 and 21 against Marek's disease in chickens.

Ligands for Toll-like receptors (TLRs) are known to stimulate immune responses, leading to protection against bacterial and viral pathogens. Here, we aimed to examine the effects of various TLR ligands on the development of Marek's disease in chickens. Specific-pathogen free chickens were treated with a series of TLR ligands that interact with TLR3, TLR9 and TLR21. In a pilot study, it was determined that TLR4 and TLR21 ligands are efficacious, in that they could reduce the incidence of Marek's disease tumors in infected birds. Hence, in a subsequent study, chickens were treated with lipopolysaccharide (LPS) as a TLR4 and CpG oligodeoxynucleotides (ODN) as TLR21 agonists before being challenged with the RB1B strain of Marek's disease virus (MDV) via the respiratory route. The results demonstrated that the administration of LPS or CpG ODN, but not PBS or non-CpG ODN, delayed disease onset and reduced MDV genome copy number in the spleens of infected chickens. Taken together, our data demonstrate that TLR4 and 21 agonists modulate anti-virus innate immunity including cytokine responses in MD-infected chicken and this response can only delay, but not inhibit, disease progression.

Expansion of anti-mesothelin specific CD4+ and CD8+ T cell responses in patients with pancreatic carcinoma.

We aimed to assess the status of naturally occurring CD4(+) and CD8(+) T cell responses to a tumour associated antigen, Mesothelin, in patients with pancreatic carcinoma and study the effects of elevated IL-10 on Mesothelin-specific T cell responses. For that sake, short term T cell lines were generated from PBMCs of 16 healthy controls, 15 patients with benign pancreatic diseases and 25 patients with pancreatic carcinoma and Mesothelin-specific CD4(+) and CD8(+) T cell responses were analysed using intracellular cytokine assays for IFN-γ. Plasma levels of IL-10 and Mesothelin were measured using cytometric bead array and ELISA assay, respectively. The blocking assays were performed to assess the effects of IL-10 on Mesothelin-specific T cell responses. Here, we demonstrate that the plasma levels of Mesothelin and IL-10 are significantly increased in patients with pancreatic carcinoma. Additionally, we found that (a) Mesothelin-specific T cell responses are significantly expanded in cancer patients (p = 0.0053), (b) the multifunctional CD4(+) T cell response is directed toward a broad repertoire of epitopes within the Mesothelin protein. (c) Mesothelin-specific CD4+ T cell response is directly inhibited by elevated IL-10 in cancer patients. These data provides evidence for the use of Mesothelin as an immunogen for tumour-specific T cell response.

A toll-like receptor 3 ligand enhances protective effects of vaccination against Marek's disease virus and hinders tumor development in chickens.

Marek's disease (MD) is caused by Marek's disease virus (MDV). Various vaccines including herpesvirus of turkeys (HVT) have been used to control this disease. However, HVT is not able to completely protect against very virulent strains of MDV. The objective of this study was to determine whether a vaccination protocol consisting of HVT and a Toll-like receptor (TLR) ligand could enhance protective efficacy of vaccination against MD. Hence, chickens were immunized with HVT and subsequently treated with synthetic double-stranded RNA polyriboinosinic polyribocytidylic [poly(I:C)], a TLR3 ligand, before or after being infected with a very virulent strain of MDV. Among the groups that were HVT-vaccinated and challenged with MDV, the lowest incidence of tumors was observed in the group that received poly(I:C) before and after MDV infection. Moreover, the groups that received a single poly(I:C) treatment either before or after MDV infection were better protected against MD tumors compared to the group that only received HVT. No association was observed between viral load, as determined by MDV genome copy number, and the reduction in tumor formation. Overall, the results presented here indicate that poly(I:C) treatment, especially when it is administered prior to and after HVT vaccination, enhances the efficacy of HVT vaccine and improves protection against MDV.

Induction of CD152 (CTLA-4) and LAP (TGF-β1) in human Foxp3- CD4+ CD25- T cells modulates TLR-4 induced TNF-α production.

CD152 (CTLA-4) is a co-stimulatory molecule that is expressed by T cells and negatively regulates immune responses. Here, we report the identification of a novel ligand, GPC(81-95), with the ability to induce both CD152 and LAP (TGF-β1) on human Foxp3(-) CD25(-) CD4(+) T cells. The results demonstrate that GPC(81-95) peptide-induced cell surface CD152 is endocytosed back into the cell during stimulation. The protein export and exocytosis of CD152 is also induced by this ligand. The inhibitory effects of GPC(81-95) on LPS-induced TNF-α production was shown to be closely associated with its ability to induce both LAP (TGF-β1) and CD152. Taken together, we have shown that a novel peptide ligand stimulates LAP (TGF-β1) and CD152 expression on resting CD4 T cells and have demonstrated that GPC(81-95) is a useful tool to study the functional properties of LAP (TGF-β1)(+) CD152(+) CD4(+) T cells.

Vaccination with CpG-adjuvanted avian influenza virosomes promotes antiviral immune responses and reduces virus shedding in chickens.

The use of virosomes as a vaccine platform has proven successful against several viruses. Here we examined the protective efficacy of a virosome-based vaccine consisting of avian influenza virus (AIV) A/Duck/Czech/56/H4N6 in chickens against a homologous AIV challenge. Virosomes adjuvanted with CpG-ODN or recombinant chicken interferon (IFN)-γ significantly reduced virus shedding after virus challenge. Furthermore, immunization with virosomes adjuvanted with CpG-ODN increased hemagglutination inhibition (HI) and virus-specific neutralizing serum antibodies, as well as virus-specific serum IgG and mucosal IgA responses. We also found a significant increase in the expression of type I and II interferon genes in the protected birds following virus challenge. In summary, this study demonstrated the ability of virosomes adjuvanted with CpG-ODN to reduce AIV shedding, and elicit virus-specific protective antibody responses in vaccinated birds.

Interferon-γ influences immunity elicited by vaccines against very virulent Marek's disease virus.

Vaccination of chickens with herpesvirus of turkey (HVT) confers only partial protection against challenge with a very virulent Marek's disease virus (MDV). Here, we evaluated the ability of recombinant chicken interferon-gamma (rChIFN-γ) to enhance protective efficacy of HVT against the very virulent MDV strain, RB1B. The bioactivity of IFN-γ expressed by a plasmid expression vector was confirmed by its ability to stimulate a chicken macrophage cell line (HD11) to produce nitric oxide (NO) in vitro. The administration of HVT with 5μg of pcDNA:chIFN-γ plasmid reduced the incidence of tumor development significantly when compared to vaccinated birds (77.7% in the HVT+empty vector group and 80% in HVT group versus 33.3% in the HVT+chIFN-γ group) and significantly increased IFN-γ expression in the splenocytes of the protected group, suggesting that rChIFN-γ increases the potency of HVT against MDV. Further analysis demonstrated that the protected birds that received HVT vaccine and/or plasmid had lower MDV genome load and lower amounts of transcripts for meq and vIL-8 than in the birds without lesions. Similarly, lower expression of IL-10, IL-18 and IL-6 was observed in the chickens without lesions compared to the chickens that had lesions, suggesting an inverse association between up-regulation of these cytokines and vaccine-induced immunity. In conclusion, IFN-γ can positively influence immunity conferred by HVT vaccination against challenge with a very virulent Marek's disease virus (vvMDV) in chickens.

Induction of latency-associated peptide (transforming growth factor-β(1)) expression on CD4+ T cells reduces Toll-like receptor 4 ligand-induced tumour necrosis factor-α production in a transforming growth factor-β-dependent manner.

CD4(+) T cells expressing the latent form of transforming growth factor-β [latency-associated peptide (LAP) (TGF-β(1))] play an important role in the modulation of immune responses. Here, we identified a novel peptide ligand (GPC(81-95) ) with an intrinsic ability to induce membrane-bound LAP (TGF-β(1)) expression on a subpopulation of human CD4(+) T cells (using flow cytometry; ranging from 0·8% to 2·6%) and stimulate peripheral blood mononuclear cells to release LAP (TGF-β(1) ) (using ELISPOT assay; ranging from 0·03% to 0·16%). In spite of this low percentage of responding cells, GPC(81-95) significantly reduced Toll-like receptor 4 ligand-induced tumour necrosis factor-α production in a TGF-β(1) - and CD4(+) T-cell-dependent manner. The results demonstrate that GPC(81-95) is a useful tool to study the functional properties of a subpopulation of LAP (TGF-β(1))(+) CD4(+) T cells and suggest a pathway that can be exploited to suppress inflammatory response.

Enhancement of immunogenicity of a virosome-based avian influenza vaccine in chickens by incorporating CpG-ODN.

Influenza virosomes are virus-like particles, representing a platform for vaccine development. In this study, we examined the immunogenicity of avian influenza virosomes with or without inclusion of recombinant chicken interferon-gamma (rChIFN-γ) or CpG-ODN in chickens. Immunization with virosomes adjuvanted with CpG-ODN elicited the highest haemagglutination inhibition antibody titres, as well as IgG and IgA serum antibody responses. Moreover, Virosomes+CpG-ODN formulation induced an antigen-specific spleen cell proliferation and IFN-γ expression. In conclusion, our results demonstrated that virus-specific antibody- and cell-mediated responses may be induced in chickens immunized with virosomes and these responses can be enhanced by incorporating CpG-ODN in the virosome vaccine formulation.

Alpha-fetoprotein specific CD4 and CD8 T cell responses in patients with hepatocellular carcinoma.

The presence of CD8 T cell responses to tumor associated antigens have been reported in patients with different malignancies. However, there is very little information on a comparable CD8 and CD4 T cell response to a tumor antigen in liver cancer patients. Here, we re-examine the kinetic and the pattern of T helper 1 and cytotoxic T lymphocyte responses to alpha-fetoprotein (AFP), a tumor rejection antigen in hepatocellular carcinoma (HCC). Then, we discuss the possibility of using AFP-based immunotherapy in combination with necrotizing treatments in HCC patients.

Expression profiling of genes associated with regulatory functions of T-cell subsets in Marek's disease virus-infected chickens.

The environment of tumours caused by Marek's disease virus (MDV) in chickens has been shown to have an immunoregulatory phenotype. The objective of the present study was to examine the expression of key T-regulatory markers during various stages of MDV pathogenesis. Specific-pathogen free (SPF) as well as major histocompatibility complex-defined chickens were infected with the RB1B and JM-16 strains of MDV, respectively. CD4(+) and CD8(+) T cells from the spleens of infected as well as age-matched controls were sorted by flow cytometry at 4, 10, and 21 days post infection (d.p.i.). The expression of molecules such as CTLA-4, IL-2aR (CD25), PD-1 and PDL-1 was quantified by real-time, quantitative, reverse-transcription polymerase chain reaction. There was an up-regulation of CTLA-4 in CD4(+) T cells at 4 d.p.i. The expression of PD-1 was also up-regulated in the CD4(+) T-cell subset of SPF birds at 21 d.p.i. Furthermore, the expression of PD-1 was enhanced in CD4(+) and CD8(+) T cells of genetically susceptible chickens, linking this molecule to susceptibility to disease. The expression of CD25 was down-regulated in both SPF and genetically defined birds after infection. This may be a mechanism through which the virus exerts its immunosuppressive effects. In conclusion, the results of the present study provide more insight into immunomodulatory processes that occur in the lymphoid tissues of MDV-infected chickens.

Insights into the role of Toll-like receptors in modulation of T cell responses.

The innate immune receptors, such as Toll-like receptors (TLRs), are intimately involved in the early sensing of invading microorganisms or their structural components. Engagement of TLRs with their ligands results in activation of several downstream intracellular pathways leading to activation of innate and adaptive immune system cells. It was initially thought that TLRs are primarily expressed by antigen-presenting cells (APCs), such as macrophages and dendritic cells, and that interactions between microbial ligands and TLRs in these cells will indirectly result in activation of cells of the adaptive immune system, especially T cells. However, it has now become evident that TLRs are also expressed by various T cell subsets, such as conventional αβT cells, regulatory T cells, and γδT cells as well as natural killer T cells. Importantly, it appears that at least in some of these T cell subsets, TLRs are functionally active, because stimulation of these cells with TLR agonists in the absence of APCs results in exertion of effector or regulatory functions of T cells. The present review attempts to summarize the recent findings related to TLR expression in different T cell subsets and the direct role of TLRs in the induction and regulation of T cell responses, including those responses that occur at mucosal surfaces. In addition, the potential use of TLR agonists for steering T cell responses as a prophylactic or therapeutic strategy in the context of infectious, allergic or autoimmune diseases is explored.

Characterization of host responses against a recombinant fowlpox virus-vectored vaccine expressing the hemagglutinin antigen of an avian influenza virus.

There currently are commercial fowlpox virus (FPV)-vectored vaccines for use in chickens, including TROVAC-AIV H5, which expresses the hemagglutinin (HA) antigen of an avian influenza virus and can confer immunity against avian influenza in chickens. Despite the use of recombinant FPV (rFPV) for vaccine delivery, very little is known about the immune responses generated by these viruses in chickens. The present study was designed to investigate host responses to rFPV in vivo and in vitro. In cultured cells infected with TROVAC-AIV H5, there was an early increase in the expression of type I interferons (IFN), Toll-like receptors 3 and 7 (TLR3 and TLR7, respectively), TRIF, and MyD88, which was followed by a decrease in the expression of these genes at later time points. There also was an increase in the expression of interleukin-1beta (IL-1beta), IL-8, and beta-defensin genes at early time points postinfection. In chickens immunized with TROVAC-AIV H5, there was higher expression of IFN-gamma and IL-10 at day 5 postvaccination in spleen of vaccinated birds than in that of control birds. We further investigated the ability of the vaccine to induce immune responses against the HA antigen and discovered that there was a cell-mediated response elicited in vaccinated chickens against this antigen. The findings of this study demonstrate that FPV-vectored vaccines can elicit a repertoire of responses marked by the early expression of TLRs, type I interferons, and proinflammatory cytokines, as well as cytokines associated with adaptive immune responses. This study provides a platform for designing future generations of rFPV-vectored vaccines.

Cell-mediated immune responses to alpha-fetoprotein and other antigens in hepatocellular carcinoma.

Cell-mediated immune responses play an important role in the control of tumour growth. CD4 and CD8 T cells recognise tumour antigens presented via major histocompatibility complex molecules of antigen presenting cells and develop into effector cells with the ability to identify and kill tumour cells. Here, we re-examine the adaptive immune response to tumour antigens expressed by hepatocellular carcinoma (HCC) and discuss approaches that could be applied in future T-cell-based immunotherapy schedules to induce a potent and effective antitumour immunity. Moreover, we discuss cytotoxic T lymphocyte and Th1 responses to tumour antigens in patients with HCC and evaluate the effects of conventional treatments on antitumour T-cell responses.

Identification of a dual-specific T cell epitope of the hemagglutinin antigen of an h5 avian influenza virus in chickens.

Avian influenza viruses (AIV) of the H5N1 subtype have caused morbidity and mortality in humans. Although some migratory birds constitute the natural reservoir for this virus, chickens may play a role in transmission of the virus to humans. Despite the importance of avian species in transmission of AIV H5N1 to humans, very little is known about host immune system interactions with this virus in these species. The objective of the present study was to identify putative T cell epitopes of the hemagglutinin (HA) antigen of an H5 AIV in chickens. Using an overlapping peptide library covering the HA protein, we identified a 15-mer peptide, H5(246-260,) within the HA1 domain which induced activation of T cells in chickens immunized against the HA antigen of an H5 virus. Furthermore, H5(246-260) epitope was found to be presented by both major histocompatibility complex (MHC) class I and II molecules, leading to activation of CD4+ and CD8+ T cell subsets, marked by proliferation and expression of interferon (IFN)-gamma by both of these cell subsets as well as the expression of granzyme A by CD8+ T cells. This is the first report of a T cell epitope of AIV recognized by chicken T cells. Furthermore, this study extends the previous finding of the existence of dual-specific epitopes in other species to chickens. Taken together, these results elucidate some of the mechanisms of immune response to AIV in chickens and provide a platform for creation of rational vaccines against AIV in this species.

Human CD4(+) T cells recognize an epitope within alpha-fetoprotein sequence and develop into TGF-beta-producing CD4(+) T cells.

There is limited information on the influence of tumor growth on the expansion of tumor-specific TGF-beta-producing CD4(+) T cells in humans. alpha-Fetoprotein (AFP) is an oncofetal Ag and has intrinsic immunoregulatory properties. In this study, we report the identification and characterization of subsets of CD4(+) T cells that recognize an epitope within the AFP sequence (AFP(46-55)) and develop into TGF-beta-producing CD4(+) T cells. In a peptide-specific and dose-dependent manner, AFP(46-55) CD4(+) T cells produce TGF-beta, GM-CSF, and IL-2 but not Th1-, Th2-, Th17-, or Tr1-type cytokines. These cells express CTLA-4 and glucocorticoid-induced TNR receptor and inhibit T cell proliferation in a contact-dependent manner. In this study, we show that the frequency of AFP(46-55) CD4(+) T cells is significantly higher (p = 001) in patients with hepatocellular carcinoma than in healthy donors, suggesting that these cells are expanded in response to tumor Ag. In contrast, tumor necrosis-inducing treatments that are shown to improve survival rate can shift the Th1/TGF-beta-producing CD4(+) T cell balance in favor of Th1 responses. Our data demonstrate that tumor Ags may contain epitopes which activate the expansion of inducible regulatory T cells, leading to evasion of tumor control.

Unmasking of alpha-fetoprotein-specific CD4(+) T cell responses in hepatocellular carcinoma patients undergoing embolization.

Necrosis of tumor cells can activate both innate and adaptive antitumor immunity. However, there is little information on the effects of necrosis-inducing cancer treatments on tumor-specific T cell immune responses in humans. We studied the effects of a necrosis-inducing treatment (embolization) on anti-alpha-fetoprotein (AFP)-specific CD4(+) T cell responses in hepatocellular carcinoma (HCC) patients and controls using an array of AFP-derived peptides. In this study, we show that AFP-specific CD4(+) T cell responses to three immunodominant epitopes in HCC patients were significantly expanded during (p < 0.0001) and after embolization (p < 0.002). The development of higher frequencies of AFP-specific CD4(+) T cells after treatment were significantly associated with the induction of >50% necrosis of tumor and an improved clinical outcome (p < 0.007). In addition, we identified two novel HLA-DR-restricted AFP-derived CD4(+) T cell epitopes (AFP(137-145) and AFP(249-258)) and showed that the CD4(+) T cells recognizing these epitopes produce Th1 (IFN-gamma and TNF-alpha) but not Th2 (IL-5)-type cytokines. AFP(137-145)-, AFP(249-258)-, and AFP(364-373)-specific CD4(+) T cells were detected in HCC patients but not in patients with chronic liver diseases or healthy donors. In conclusion; our study shows that induction of tumor necrosis by a conventional cancer treatment can unmask tumor rejection Ag cell-mediated immunity and provides a rationale for combining embolization with immunotherapy in HCC patients.

Analysis of CD4+ T-Cell responses to a novel alpha-fetoprotein-derived epitope in hepatocellular carcinoma patients.

Alpha-fetoprotein (AFP) is a tumor-associated antigen in hepatocellular carcinoma and is a target for the development of cancer vaccine. Four immunodominant AFP-derived HLA-A*0201-restricted peptides have been identified and the administration of these peptides with an adjuvant has stimulated AFP-specific CTL responses in hepatocellular carcinoma patients. However, no AFP-derived CD4 T-cell epitope has yet been reported and the status of AFP-specific CD4(+) T-cell responses in hepatocellular carcinoma patients is not fully understood. The aim of this study was to analyze naturally occurring CD4(+) T-cell responses to AFP.

Direct processing and presentation of antigen from malaria sporozoites by professional antigen-presenting cells in the induction of CD8 T-cell responses.

Irradiated malaria sporozoites induce better protection than viable untreated sporozoites. We observed early differences between irradiated and viable untreated sporozoites in priming responses in vivo to a protective CD8 T-cell epitope, pb9, of the circumsporozoite protein of Plasmodium berghei. Sporozoites were processed for MHC class I presentation by dendritic cells (DC) to prime pb9-specific IFN-gamma-producing CD8 T cells. DC pulsed with untreated and irradiated sporozoites were similarly capable of priming central memory T-cell responses, detectable by the IFN-gamma cultured ELISPOT assay. However, irradiation significantly enhanced sporozoites' ability to prime effector T-cell responses detectable by the IFN-gammaex vivo ELISPOT assay. Irradiation also enhanced the ability of splenic APC to process and present sporozoites in order to re-stimulate pb9-specific polyclonal and clonal T-cell responses. Sporozoites did not stimulate T cells in the absence of APC. Over-irradiation decreased the sporozoites' T-cell stimulating capacity in vitro at high parasite doses, which may indicate that an optimal irradiation dose is necessary to induce protective immunity by sporozoite inoculation. The induction of sporozoite-specific CD8 T-cell responses without the need for liver stage infection identifies a potentially important mechanism in the development of pre-erythrocytic immunity.

Dendritic cells infected by recombinant modified vaccinia virus Ankara retain immunogenicity in vivo despite in vitro dysfunction.

The administration of recombinant vaccinia virus Ankara (MVA) encoding a CTL epitope (pb9) from a malaria antigen induced activation and maturation of splenic dendritic cells (DCs) in vivo. In contrast, incubation of immature dendritic cells (iDCs) with the MVA, in vitro, resulted in down-regulation of MHC class I molecules and reduced their T-cell stimulatory ability. However, the ability of the infected DC to induce an antigen-specific CTL response, in vivo, remained intact. Furthermore, the administration of recombinant MVA-infected DC, but not pb9 peptide-pulsed DC, boosted and expanded the anti-pb9 CTL response that was primed by pb9 peptide-pulsed DC. These data indicate that despite the ability of poxviruses to impair DC maturation in vivo, the important ability of MVA to boost CD8 T-cell response in vivo is mediated at the level of the infected dendritic cells.

Alpha-fetoprotein impairs APC function and induces their apoptosis.

alpha-Fetoprotein (AFP) is a tumor-associated Ag, and its serum level is elevated in patients with hepatocellular carcinoma (HCC). In vitro, AFP induces functional impairment of dendritic cells (DCs). This was demonstrated by the down-regulation of CD40 and CD86 molecules and the impairment of allostimulatory function. Also, AFP was found to induce significant apoptosis of DCs, and AFP-treated DCs produced low levels of IL-12 and TNF-alpha, a cytokine pattern that could hamper an efficient antitumor immune response. Ex vivo, APCs of patients with HCC and high levels of AFP produced lower levels of TNF-alpha than that of healthy individuals. In conclusion, these results illustrate that AFP induces dysfunction and apoptosis of APCs, thereby offering a mechanism by which HCC escapes immunological control.

Splenic dendritic cell subsets prime and boost CD8 T cells and are involved in the generation of effector CD8 T cells.

The ability of the dendritic cell (DC) subsets, CD8alpha+ and CD8alpha- DCs, to initiate a CD8 T cell response or to activate memory CD8 T cells and generate effector CD8 T cells has been controversial. In this study, we analyse the capacity of splenic DC subsets to induce CD8 T cell responses to a CD8 T cell epitope (pb9) of a malaria antigen. The administration of peptide-pulsed CD8alpha- or CD8alpha+ DCs primes and boosts a primed CD8 T cell response against the malaria epitope. In vitro, depletion of CD11c(+) DCs from mouse splenocytes, immunised with recombinant vaccinia virus Ankara (MVA) expressing pb9 epitope, significantly reduced the generation of pb9-specific IFNgamma producing effector CD8 T cells, indicating that splenic DCs are involved in the development of pb9-specific IFNgamma producing effector cells. Taken together, this result shows that both DC subsets have the ability to prime and boost CD8 T cell responses and are involved in the activation of memory CD8 T cells.

Tracking the source of the hepatitis B virus-specific CD8 T cells during lamivudine treatment.

Lamivudine treatment in chronic hepatitis B leads to the reconstitution of virus-specific T cells in the circulation, but it is not clear whether this is the preferential result of T cell efflux from the liver or lymph nodes. To address this question, the frequency and function of liver-, lymph node-, and blood-derived hepatitis B virus (HBV)-specific CD8 T cells were analyzed in patients treated with lamivudine and undergoing liver transplantation. HBV-specific CD8 T cells, identified in portal lymph nodes, were able to expand in vitro after antigen-specific stimulation and displayed a heterogeneous profile of cytokine production. These findings suggest that the peripherally reconstituted HBV-specific CD8 T cells can originate from precursor cells within lymph nodes.

Protective immunity against Mycobacterium tuberculosis induced by dendritic cells pulsed with both CD8(+)- and CD4(+)-T-cell epitopes from antigen 85A.

Immunization with DNA followed by modified vaccinia virus Ankara strain, both expressing the antigen 85A, induced both CD4(+)- and CD8(+)-T-cell responses in BALB/c mice. Following challenge with Mycobacterium tuberculosis, this prime-boost regimen produced protection equivalent to that conferred by Mycobacterium bovis BCG. Following immunization with dendritic cells pulsed with an antigen 85A CD4(+)- or CD8(+)-restricted epitope, alone or in combination, copresentation of both epitopes on the same dendritic cell was required for protection, demonstrating that induced CD8(+) T cells can play a protective role against tuberculosis.