Biomedical subjects

Weisan Chen

Publications and source records attributed to Weisan Chen.

At least 19 recordsLinked to original sources

PubMed2006

Immunoproteasome subunit deficiencies impact differentially on two immunodominant influenza virus-specific CD8+ T cell responses.

Primary CD8+ T cell (T(CD8+)) responses to viruses are directed toward multiple Ags and shaped by both the level of Ag presentation and the underlying Ag-specific T(CD8+) repertoire. The relative importance of these factors in deciding the hierarchy of T(CD8+) responses and how they are influenced by the immunoproteasome are not well understood. Using an influenza infection model in mice deficient in various immunoproteasome subunits, we observe that Ag presentation and T(CD8+) repertoire are altered in an epitope-specific and immunoproteasome subunit-dependent manner. More importantly, we find that the level of Ag presentation and the extent of the underlying repertoire can work either alone or in concert to determine definitively the magnitude of the individual T(CD8+) responses and hence the overall T(CD8+) hierarchy. Together, these results provide a clearer understanding of how immunodominance hierarchies are established.

Animals↗

PubMed2006

Striking immunodominance hierarchy of naturally occurring CD8+ and CD4+ T cell responses to tumor antigen NY-ESO-1.

Immunodominance has been well-demonstrated in many antiviral and antibacterial systems, but much less so in the setting of immune responses against cancer. Tumor Ag-specific CD8+ T cells keep cancer cells in check via immunosurveillance and shape tumor development through immunoediting. Because most tumor Ags are self Ags, the breadth and depth of antitumor immune responses have not been well-appreciated. To design and develop antitumor vaccines, it is important to understand the immunodominance hierarchy and its underlying mechanisms, and to identify the most immunodominant tumor Ag-specific T cells. We have comprehensively analyzed spontaneous cellular immune responses of one individual and show that multiple tumor Ags are targeted by the patient's immune system, especially the "cancer-testis" tumor Ag NY-ESO-1. The pattern of anti-NY-ESO-1 T cell responses in this patient closely resembles the classical broad yet hierarchical antiviral immunity and was confirmed in a second subject.

Animals↗

PubMed2006

Dynamic quantification of MHC class I-peptide presentation to CD8+ T cells via intracellular cytokine staining.

In order to further our basic understanding of antigen processing and presentation as well as to translate that knowledge into clinically effective vaccines and immunotherapies, having appropriate tools to study MHC class I-peptide presentation is highly desirable. Current methods are based upon HPLC fractionation of extracted peptides, monoclonal Ab, multivalent T cell receptors (TCR), T cell hybridomas, TCR transgenic cells, and T cell lines. However, each of these is associated with problems that make them either difficult to apply generally or too insensitive to adequately quantitate antigen presentation. We have developed a method based upon intracellular cytokine staining (ICS) that dynamically and relatively quantitates MHC class I-peptide presentation to CD8+ T cells in a manner that is both widely applicable and highly sensitive. It is well-suited to assess antigen presentation in its early stages, does not require fixation nor labeling of antigen presenting cells (APC), can be used to examine cross-presentation, and is able to directly employ ex vivo T cells which obviates the need for the development and maintenance of T cell lines and hybridomas. Our method represents a simple yet powerful tool that others interested in studying antigen processing and presentation should find of great practical value.

Animals↗

PubMed2006

A virus-specific CD8+ T cell immunodominance hierarchy determined by antigen dose and precursor frequencies.

Immunodominance hierarchies are a substantial, but poorly understood, characteristic of CD8(+) T cell-mediated immunity. Factors influencing the differential responses to the influenza A virus nucleoprotein (NP(366-374)) and acid polymerase (PA(224-233)) peptides presented by H2D(b) have been analyzed by disabling (N5--> Q substitution) these peptides in their native configuration, then expressing them in the viral neuraminidase protein. This strategy of shifting epitopes within the same viral context resulted in an apparent equalization of D(b)NP(366) [epitope consisting of viral nucleoprotein (NP) amino acid residues 366-374 complexed with the H2D(b) MHC class I glycoprotein] and D(b)PA(224) (H2D(b)+PA(224-233)) epitope abundance after direct infection in vitro and induced reproducible changes in the magnitude of the D(b)NP(366)- and D(b)PA(224)-specific T cell subsets generated after infection of mice. Comparison of D(b)NP(366)- and D(b) PA(224)-specific CD8(+) T cell responses induced from the native configuration and from the viral neuraminidase stalk demonstrated that the size of both primary and secondary responses is influenced by relative epitope levels and that, at least after secondary challenge, the magnitude of responses is also determined by CD8(+) T cell precursor frequency. Thus, this immunodominance hierarchy is a direct function of antigen dose and T cell numbers.

Animals↗

PubMed2006

Immunodominance and immunodomination: critical factors in developing effective CD8+ T-cell-based cancer vaccines.

The focusing of cellular immunity toward one, or just a few, antigenic determinant, even during immune responses to complex microorganisms or antigens, is known as immunodominance. Although described in many systems, the mechanisms of determinant immunodominance are only just beginning to be appreciated, especially in relation to the interplay between T cells of differing specificities and the interactions between T cells and the antigen-presenting cells (APCs). The outcome of these cellular interactions can lead to a form of immune suppression of one specificity by another-described as "immunodomination". The specific and detailed mechanisms involved in this process are now partly defined. A full understanding of all the factors that control immunodominance and influence immunodomination will help us to develop better viral and cancer vaccines.

Animals↗

PubMed2006

Directions in the immune targeting of cancer: lessons learned from the cancer-testis Ag NY-ESO-1.

Since the early 1990s, numerous cancer Ag have been defined and for a handful of these there is now some clinical experience, which has made it possible to assess their value as targets for cancer immunotherapy. The cancer-testis Ag have been particularly attractive because their expression is limited to cancer and virtually no non-malignant cells apart from germ cells and trophoblast. Among these, NY-ESO-1 has been the focus of our attention. The exceptional immunogenicity of this Ag coupled with its widespread distribution among many cancer types make it a very good vaccine candidate, with the potential to be used in vaccines against many types of malignancies. This article reviews emerging knowledge about the biology of NY-ESO-1 and experience with the early clinical development of vaccines directed against NY-ESO-1. These early studies have yielded a wealth of information about the immunology of NY-ESO-1 and set the scene for future clinical strategies for immune targeting of cancer.

Antigens, Neoplasm↗

PubMed2005

T cell determinants incorporating beta-amino acid residues are protease resistant and remain immunogenic in vivo.

A major hurdle in designing successful epitope-based vaccines resides in the delivery, stability, and immunogenicity of the peptide immunogen. The short-lived nature of unmodified peptide-based vaccines in vivo limits their therapeutic application in the immunotherapy of cancers and chronic viral infections as well as their use in generating prophylactic immunity. The incorporation of beta-amino acids into peptides decreases proteolysis, yet its potential application in the rational design of T cell mimotopes is poorly understood. To address this, we have replaced each residue of the SIINFEKL epitope individually with the corresponding beta-amino acid and examined the resultant efficacy of these mimotopes. Some analogs displayed similar MHC binding and superior protease stability compared with the native epitope. Importantly, these analogs were able to generate cross-reactive CTLs in vivo that were capable of lysing tumor cells that expressed the unmodified epitope as a surrogate tumor Ag. Structural analysis of peptides in which anchor residues were substituted with beta-amino acids revealed the basis for enhanced MHC binding and retention of immunogenicity observed for these analogs and paves the way for future vaccine design using beta-amino acids. We conclude that the rational incorporation of beta-amino acids into T cell determinants is a powerful alternative to the traditional homologous substitution of randomly chosen naturally occurring alpha-amino acids, and these mimotopes may prove particularly useful for inclusion in epitope-based vaccines.

Amino Acid Sequence↗

PubMed2005

Characterization of antigen-specific CD8+ T lymphocyte responses in skin and peripheral blood following intradermal peptide vaccination.

Immune responses to cancer vaccines are commonly tested by measuring cutaneous reactions to intradermal (i.d.) antigen. When well-characterized peptide epitopes are injected i.d., infiltrates of CD4+ and CD8+ T lymphocytes are frequently seen. In this study, we have further characterized T cells derived from vaccine-infiltrating lymphocyte (VIL) responses. We found that the infiltrates capable of producing IFN-gamma and cytolytic activity could recognize vaccine peptide, as well as antigen-positive melanoma cells. We studied antigen-specific T cell responses from VILs and peripheral blood in 10 patients who participated in a clinical trial. All patients received systemic Flt3 ligand (20 microg/kg/d) and i.d. peptides: Three NY-ESO-1 peptides, SLLMWITQCFL (157-167), SLLMWITQC (157-165), QLSLLMWIT (155-163); tyrosinase internal peptide YMDGTMSQV (368-376); Melan-A/MART-1 analogue peptide ELAGIGILTV (26-35, E27L substitution); and influenza matrix peptide GILGFVFTL (58-66). In 54 paired VIL and peripheral blood analyses, a good correlation was found between responses in skin and in blood. These cells could be rapidly expanded in a short-term assay and thus appear to be memory T cells. The demonstrated presence of antigen-specific T cells at vaccination sites validates this method of assessing the immune response to i.d. vaccines.

Antigens, Neoplasm↗

PubMed2005

RNAdb--a comprehensive mammalian noncoding RNA database.

In recent years, there have been increasing numbers of transcripts identified that do not encode proteins, many of which are developmentally regulated and appear to have regulatory functions. Here, we describe the construction of a comprehensive mammalian noncoding RNA database (RNAdb) which contains over 800 unique experimentally studied non-coding RNAs (ncRNAs), including many associated with diseases and/or developmental processes. The database is available at http://research.imb.uq.edu.au/RNAdb and is searchable by many criteria. It includes microRNAs and snoRNAs, but not infrastructural RNAs, such as rRNAs and tRNAs, which are catalogued elsewhere. The database also includes over 1100 putative antisense ncRNAs and almost 20,000 putative ncRNAs identified in high-quality murine and human cDNA libraries, with more to be added in the near future. Many of these RNAs are large, and many are spliced, some alternatively. The database will be useful as a foundation for the emerging field of RNomics and the characterization of the roles of ncRNAs in mammalian gene expression and regulation.

Animals↗

PubMed2004

Tumor antigen processing and presentation depend critically on dendritic cell type and the mode of antigen delivery.

Dendritic cells (DCs) are being evaluated for cancer immunotherapy due to their unique ability to induce tumor-directed T-cell responses. Here we report that the type of human DC, the mode of activation, and the strategy for delivery of antigen are 3 critical factors for efficient stimulation of tumor-specific CD8+ and CD4+ T cells. Only CD1c+ blood DCs and monocyte-derived DCs (MoDCs) were capable of presenting epitopes of the full-length tumor antigen NY-ESO-1 on both major histocompatibility complex (MHC) class I (cross-presentation) and MHC II, whereas plasmacytoid DCs were limited to MHC II presentation. Cross-presentation was inefficient for soluble protein, but highly efficient for antigen-antibody immune complexes (NY-ESO-1/IC) and for protein formulated with ISCOMATRIX adjuvant (NY-ESO-1/IMX). DC activation with CD40L further enhanced cross-presentation efficiency. The mode of antigen delivery was found to be a determining factor for cytosolic proteolysis by DCs. Immune complexes (ICs) targeted a slow, proteasome-dependent cross-presentation pathway, whereas ISCOMATRIX (IMX) targeted a fast, proteasome-independent pathway. Both cross-presentation pathways resulted in a long-lived, T-cell stimulatory capacity, which was maintained for several days longer than for DCs pulsed with peptide. This may provide DCs with ample opportunities for sensitizing tumor-specific T cells against a broad array of tumor antigen epitopes in lymph nodes.

Adjuvants, Immunologic↗

PubMed2004

Reversal in the immunodominance hierarchy in secondary CD8+ T cell responses to influenza A virus: roles for cross-presentation and lysis-independent immunodomination.

Immunodominance is a central feature of CD8+ T cell (TCD8+) responses to pathogens, transplants, and tumors. Determinants occupy a stable position in an immunodominance hierarchy (alpha-, beta-, etc.) defined by the frequencies of responding TCD8+. In this paper, we study the mechanistic basis for place-swapping between alpha- (acid polymerase (PA)(224-233)) and beta-determinants (nuclear protein 366-374) in primary vs secondary anti-influenza A virus (IAV) responses in mice. This phenomena was recently correlated with the inability of IAV-infected nondendritic cells (DCs) to generate PA(224-233), and it was proposed that secondary TCD8+ are principally activated by IAV-infected epithelial cells, while primary TCD8+ are activated by IAV-infected DCs. In this study, we show that the inability of non-DCs to generate PA(224-232) is relative rather than absolute, and that the preferential use of cross-priming in secondary anti-IAV responses can also account for the revised hierarchy. We further show that immunodomination of PA(224-233)-specific TCD8+ by nucleoprotein 366-374-specific TCD8+ plays a critical role in the phenomena, and that this is unlikely to be mediated by TCD8+ lysis of APCs or other cells.

Adoptive Transfer↗

PubMed2004

The impact of imiquimod, a Toll-like receptor-7 ligand (TLR7L), on the immunogenicity of melanoma peptide vaccination with adjuvant Flt3 ligand.

Dendritic cells (DCs) show promise as adjuvants in anticancer immunotherapeutic strategies. Flt3 ligand (FL) is a hematopoietic growth factor that increases the number of immature DCs in the blood and other tissues. We treated 27 patients with metastatic or high-risk resected melanoma with s.c. FL daily for 14 d in three 28 d cycles. Eighteen of these patients also received vaccination with influenza (Flu), Melan-A (Mel), tyrosinase (Tyr), and NY-ESO-1 peptides. To induce local DC maturation, 8 of the vaccinated patients had imiquimod, a Toll-like receptor-7 ligand (TLR7L), applied topically to their vaccine sites. Patients were monitored for clinical and hematological effects. Immune responses were assessed by cutaneous reactivity to vaccination and by the induction of peptide-specific CD8+ T-cells. Eight patients did not complete the protocol due to adverse events related to their cancer. The treatment was generally safe and well tolerated, although some patients developed clinically significant toxicities related to FL. FL induced increases in immature CD11c+ and CD123+ peripheral blood (PB) DCs. Other hematological effects included monocytosis, granulocytosis, and thrombocytosis, which were marked in some patients. Cutaneous reactions to peptide vaccination and circulating peptide-specific CD8+ T-cells were more frequent in imiquimod-treated patients. FL treatment of melanoma patients has pleiotropic clinical and hematological effects. In vivo maturation of FL-generated DCs using imiquimod may increase immune responses to tumor antigens.

Adjuvants, Immunologic↗

PubMed2004

Recombinant NY-ESO-1 protein with ISCOMATRIX adjuvant induces broad integrated antibody and CD4(+) and CD8(+) T cell responses in humans.

NY-ESO-1 is a "cancer-testis" antigen expressed in many cancers. ISCOMATRIX is a saponin-based adjuvant that induces antibody and T cell responses. We performed a placebo-controlled clinical trial evaluating the safety and immunogenicity of recombinant NY-ESO-1 protein with ISCOMATRIX adjuvant. Forty-six evaluable patients with resected NY-ESO-1-positive tumors received three doses of vaccine intramuscularly at monthly intervals. The vaccine was well tolerated. We observed high-titer antibody responses, strong delayed-type hypersensitivity reactions, and circulating CD8(+) and CD4(+) T cells specific for a broad range of NY-ESO-1 epitopes, including known and previously unknown epitopes. In an unplanned analysis, vaccinated patients appeared to have superior clinical outcomes to those treated with placebo or protein alone. The vaccine is safe and highly potent immunologically.

Adjuvants, Immunologic↗

PubMed2004

Immunodominant CD4+ responses identified in a patient vaccinated with full-length NY-ESO-1 formulated with ISCOMATRIX adjuvant.

There is increasing evidence showing the involvement of CD4(+) T cells in initiating and maintaining antitumor immune responses. NY-ESO-1 is expressed by various tumors but not normal tissues except testis. We conducted a cancer clinical trial by using full-length NY-ESO-1 protein formulated with ISCOMATRIX adjuvant and injected into patients intramuscularly. Autologous dendritic cells pulsed with NY-ESO-1 ISCOMATRIX in combination with overlapping synthetic peptides were used to identify immunodominant T cells from a vaccinated patient. We show here the identification and characterization of two novel CD4(+) T cell epitopes. T cells specific to these epitopes not only recognized autologous dendritic cells loaded with NY-ESO-1 but also NY-ESO-1-expressing tumor cell lines treated with IFN-gamma. One of the two responses identified was greater than the previously identified immunodominant HLA-DP4-restricted response and correlated with NY-ESO-1-specific CD8(+) T cell induction after vaccination. This T cell response was vaccinated in most patients who expressed HLA-DR2. This study has systematically surveyed patients vaccinated with full-length tumor antigen for a vaccinated CD4 helper T cell response.

Amino Acid Sequence↗

PubMed2004

NY-ESO-1 protein formulated in ISCOMATRIX adjuvant is a potent anticancer vaccine inducing both humoral and CD8+ t-cell-mediated immunity and protection against NY-ESO-1+ tumors.

NY-ESO-1 is a 180 amino-acid human tumor antigen expressed by many different tumor types and belongs to the family of "cancer-testis" antigens. In humans, NY-ESO-1 is one of the most immunogenic tumor antigens and NY-ESO-1 peptides have been shown to induce NY-ESO-1-specific CD8(+) CTLs capable of altering the natural course of NY-ESO-1-expressing tumors in cancer patients. Here we describe the preclinical immunogenicity and efficacy of NY-ESO-1 protein formulated with the ISCOMATRIX adjuvant (NY-ESO-1 vaccine). In vitro, the NY-ESO-1 vaccine was readily taken up by human monocyte-derived dendritic cells, and on maturation, these human monocyte-derived dendritic cells efficiently cross-presented HLA-A2-restricted epitopes to NY-ESO-1-specific CD8(+) T cells. In addition, epitopes of NY-ESO-1 protein were also presented on MHC class II molecules to NY-ESO-1-specific CD4(+) T cells. The NY-ESO-1 vaccine induced strong NY-ESO-1-specific IFN-gamma and IgG2a responses in C57BL/6 mice. Furthermore, the NY-ESO-1 vaccine induced NY-ESO-1-specific CD8(+) CTLs in HLA-A2 transgenic mice that were capable of lysing human HLA-A2(+) NY-ESO-1(+) tumor cells. Finally, C57BL/6 mice, immunized with the NY-ESO-1 vaccine, were protected against challenge with a B16 melanoma cell line expressing NY-ESO-1. These data illustrate that the NY-ESO-1 vaccine represents a potent therapeutic anticancer vaccine.

Adjuvants, Immunologic↗

PubMed2004

Discordant regulation of granzyme H and granzyme B expression in human lymphocytes.

We analyzed the expression of granzyme H in human blood leukocytes, using a novel monoclonal antibody raised against recombinant granzyme H. 33-kDa granzyme H was easily detected in unfractionated peripheral blood mononuclear cells, due to its high constitutive expression in CD3(-)CD56(+) natural killer (NK) cells, whereas granzyme B was less abundant. The NK lymphoma cell lines, YT and Lopez, also expressed high granzyme H levels. Unstimulated CD4(+) and particularly CD8(+) T cells expressed far lower levels of granzyme H than NK cells, and various agents that classically induce T cell activation, proliferation, and enhanced granzyme B expression failed to induce granzyme H expression in T cells. Also, granzyme H was not detected in NK T cells, monocytes, or neutrophils. There was a good correlation between mRNA and protein expression in cells that synthesize both granzymes B and H, suggesting that gzmH gene transcription is regulated similarly to gzmB. Overall, our data indicate that although the gzmB and gzmH genes are tightly linked, expression of the proteins is quite discordant in T and NK cells. The finding that granzyme H is frequently more abundant than granzyme B in NK cells is consistent with a role for granzyme H in complementing the pro-apoptotic function of granzyme B in human NK cells.

Animals↗

PubMed2004

Functional and structural characteristics of NY-ESO-1-related HLA A2-restricted epitopes and the design of a novel immunogenic analogue.

NY-ESO-1, a commonly expressed tumor antigen of the cancer-testis family, is expressed by a wide range of tumors but not found in normal adult somatic tissue, making it an ideal cancer vaccine candidate. Peptides derived from NY-ESO-1 have shown preclinical and clinical trial promise; however, biochemical features of these peptides have complicated their formulation and led to heterogeneous immune responses. We have taken a rational approach to engineer an HLA A2-restricted NY-ESO-1-derived T cell epitope with improved formulation and immunogenicity to the wild type peptide. To accomplish this, we have solved the x-ray crystallographic structures of HLA A2 complexed to NY-ESO (157-165) and two analogues of this peptide in which the C-terminal cysteine residue has been substituted to alanine or serine. Substitution of cysteine by serine maintained peptide conformation yet reduced complex stability, resulting in poor cytotoxic T lymphocyte recognition. Conversely, substitution with alanine maintained complex stability and cytotoxic T lymphocyte recognition. Based on the structures of the three HLA A2 complexes, we incorporated 2-aminoisobutyric acid, an isostereomer of cysteine, into the epitope. This analogue is impervious to oxidative damage, cysteinylation, and dimerization of the peptide epitope upon formulation that is characteristic of the wild type peptide. Therefore, this approach has yielded a potential therapeutic molecule that satiates the hydrophobic F pocket of HLA A2 and exhibited superior immunogenicity relative to the wild type peptide.

Adult↗

PubMed2004

Cross-priming of CD8+ T cells by viral and tumor antigens is a robust phenomenon.

"Cross-priming" refers to the activation of naive CD8+ T cells by antigen-presenting cells that have acquired nominal antigens from another cell. The biological relevance of cross-priming of CD8+ T cells has recently been challenged (Zinkernagel, R. M., Eur. J. Immunol. 2002. 32: 2385-2392), on the basis that responses are weak or poorly quantitated, and the determinants recognized are undefined. Here we show that cross-priming is a robust process that elicits vigorous primary responses to multiple peptides in two well-defined systems. Our findings support the relevance of cross-priming in CD8+ T cell responses to viruses and tumor cells, and demonstrate that cross-priming elicits CD8+ T cells to determinants generated by the endogenous processing pathway.

Animals↗