Induction Of Antigen-specific CD8 Research Articles

An oral recombinant Salmonella enterica serovar Typhimurium mutant elicits systemic antigen-specific CD8+ T cell cytokine responses in mice

BackgroundThe induction of antigen-specific CD8+ T cell cytokine responses against an attenuated, oral recombinant Salmonella enterica serovar Typhimurium vaccine expressing a green fluorescent protein (GFP) model antigen was investigated. A GFP expression plasmid was constructed in which the gfp gene was fused in-frame with the 5' domain of the Escherichia coli β-galactosidase α-gene fragment with expression under the lac promoter. Groups of mice were orally immunized three times with the bacteria and systemic CD8+ T cell cytokine responses were evaluated.ResultsHigh level of the GFP model antigen was expressed by the recombinant Salmonella vaccine vector. Systemic GFP-specific CD8+ T cell cytokine (IFN-γ and IL-4) immune responses were detected after mice were orally vaccinated with the bacteria. It was shown that 226 net IFN-γ and 132 net IL-4 GFP-specific SFUs/10e6 splenocytes were formed in an ELISPOT assay. The level of IFN-γ produced by GFP peptide-stimulated cells was 65.2-fold above background (p < 0.05). The level of IL-4 produced by the cells was 10.4-fold above background (p < 0.05).ConclusionThese results suggested that a high expressing recombinant Salmonella vaccine given orally to mice would elicit antigen-specific CD8+ T cell responses in the spleen. Salmonella bacteria may, therefore, be used as potential mucosal vaccine vectors.

Coaxing the liver into preventing autoimmune disease in the brain

The liver has several unique immunological properties that affect T cell activation and immune regulation. Recent studies have uncovered opportunities for the treatment of genetic disease by directing expression of the functional therapeutic protein to hepatocytes. In a new study in this issue of the JCI, Lüth and colleagues demonstrate that hepatic expression of a brain protein is protective against neuroinflammatory disease in a mouse model of human MS (see the related article beginning on page 3403). Suppression of autoimmunity was dependent on transgene expression in the liver and was mediated by induction of antigen-specific CD4+CD25+Foxp3+ Tregs. These findings suggest that the introduction of antigens to the liver may have potential as a preventative or therapeutic intervention for autoimmune disease.

Efficient augmentation of a long-lasting immune responses in HIV-1 gag DNA vaccination by IL-15 plasmid boosting

Cytokines are major regulators of the immune response, and have been used as adjuvants to improve vaccine potency. In this study, we investigated the adjuvant effects of interleukin (IL)-15 on improving the immunogenicity of human immunodeficiency virus (HIV)-1 gag DNA vaccine in Balb/c mice. During a 370-day follow-up, cellular and humoral immune responses in three separate cohorts of mice were monitored. These results were exemplified through: lymphocyte proliferation, induction of antigen-specific CD8 + T lymphocytes, long-term production of specific antibodies, and proportion of differentiated memory CD8 + T cells. These data revealed that just boost of IL-15 at day 8 after co-immunization induced more homeostatic cell proliferation, augmented proliferation frequency of IFN-γ-secreting antigen-specific CD8 + T lymphocytes, maintained the long-lasting humoral immune response and promoted the turnover of memory T cell precursors into central memory T cells. Taken together, our data demonstrated that a single IL-15 boosting can enhance both the humoral and cellular immune responses of the HIV-1 gag DNA vaccination. This novel boosting strategy may facilitate the application of IL-15 as an adjuvant for HIV vaccination.

DNA Vaccines Encoding Ii-PADRE Generates Potent PADRE-specific CD4+ T-Cell Immune Responses and Enhances Vaccine Potency

DNA Vaccines Encoding Ii-PADRE Generates Potent PADRE-specific CD4+ T-Cell Immune Responses and Enhances Vaccine Potency

Dendritic cells with TGF-β1 differentiate naïve CD4+CD25−T cells into islet-protective Foxp3+regulatory T cells

CD4(+)CD25(+)Foxp3(+) regulatory T cells (T regs) are important for preventing autoimmune diabetes and are either thymic-derived (natural) or differentiated in the periphery outside the thymus (induced). Here we show that beta-cell peptide-pulsed dendritic cells (DCs) from nonobese diabetic (NOD) mice can effectively induce CD4(+)CD25(+)Foxp3(+) T cells from naïve islet-specific CD4(+)CD25(-) T cells in the presence of TGF-beta1. These induced, antigen-specific T regs maintain high levels of clonotype-specific T cell receptor expression and exert islet-specific suppression in vitro. When cotransferred with diabetogenic cells into NOD scid recipients, T regs induced with DCs and TGF-beta1 prevent the development of diabetes. Furthermore, in overtly NOD mice, these cells are able to significantly protect syngeneic islet grafts from established destructive autoimmunity. These results indicate a role for DCs in the induction of antigen-specific CD4(+)CD25(+)Foxp3(+) T cells that can inhibit fully developed autoimmunity in a nonlymphopoenic host, providing an important potential strategy for immunotherapy in patients with autoimmune diabetes.

PART V. Modulation of Antitumor Vaccine StrategiesPreclinical and Clinical Studies of Recombinant Poxvirus Vaccines for Carcinoma Therapy

Tumor-associated antigens (TAAs) are by definition either weakly immunogenic or functionally nonimmunogenic. Vaccine strategies have been designed to present TAAs to the immune system that may result in far greater activation of T cells than that occurring naturally in the host. These strategies include (1) placing the gene coding for the tumor antigen into poxvirus vectors as a transgene; (2) using diversified prime-and-boost vaccine strategies employing two different types of poxvirus vectors; (3) using T-cell costimulation; and (4) using cytokines, including GM-CSF, as biologic adjuvants. Preclinical studies have been performed comparing the effects on induction of antigen-specific CD8 and CD4 T-cell responses using recombinant poxvirus vectors containing transgenes for a TAA and costimulatory molecules B7-1, ICAM-1, and LFA-3 (designated TRICOM). Antigen-specific T-cell responses were greatest in the group receiving the CEA-TRICOM vaccines and were shown to correlate with survival. We have now completed the first clinical trials with poxvirus vectors containing TRICOM, using the TAAs PSA, CEA, and MUC-1. In addition, clinical studies combining vaccines with radiation therapy, chemotherapy, and second-line hormone therapy have provided preliminary evidence of prolongation of time to disease progression and antigen cascade postvaccination.

Infectious tolerance and the long‐term acceptance of transplanted tissue

Short courses of antibody treatment aimed at blocking the coreceptors CD4 and CD8 and/or costimulatory molecules such as CD40L are able to bring about long-term acceptance and tolerance of allogeneic transplants. This tolerant state is operational, in that potential effector cells remain but are tightly regulated through the induction of antigen-specific CD4+ regulatory T cells (Tregs). CD4+ CD25+ FoxP3+ Tregs appear to play a prominent role, although other categories of Tregs have been documented. Transforming growth factor beta (TGFbeta) has been found to play a major role in the induction of the tolerant state with therapeutic antibodies as well as promoting the induction of FoxP3+ T cells from naïve populations. The observation that Tregs can be found in tolerated grafts has led to the idea that they may interact with the grafted tissue to establish a state of acquired privilege symmetrical with a similar privileged microenvironment around antigen-presenting cells in lymphoid tissues. Dampening of aggressive immune responses by Tregs allows antigen to persist and be presented in an innocuous way to promote tolerance in new cohorts of T cells throughout the life of the tolerated graft. Regulation may operate at many stages of an immune response, even as a censor at the terminal differentiation stages of effector function.

Induction of antigen-specific CD8+ cytotoxic T cells by dendritic cells co-electroporated with a dsRNA analogue and tumor antigen mRNA

The maturation state of dendritic cells (DCs) is an important determinant for the initiation and regulation of adaptive immune responses. In this study, we wanted to assess whether functional activation of human monocyte-derived DCs can be achieved by electroporation of an activation signal in the form of double-stranded (ds) RNA and whether simultaneous electroporation of the dsRNA with tumor antigen encoding mRNA can lead to the induction of a cytotoxic T-lymphocyte (CTL) response. Electroporation of immature DCs with poly(I:C(12)U), a dsRNA analogue, resulted in phenotypic as well as functional changes, indicative of DC maturation. Co-electroporation of DCs with both poly(I:C(12)U) and Melan-A/MART-1 encoding mRNA induced strong anti-Melan-A/MART-1 CD8(+) T-cell responses in vitro. Higher numbers of Melan-A/MART-1-specific CTLs were consistently obtained with poly(I:C(12)U)-activated DCs compared to DCs matured in the presence of an inflammatory cytokine cocktail. These results indicate that DC co-electroporation with both dsRNA and tumor antigen encoding mRNA induces fully activated and antigen-loaded DCs that promote antigen-specific CTL responses and may provide the basis for future immunotherapeutic strategies.

Induction of antigen-specific CD4- and CD8-mediated T-cell responses by fusions of autologous dendritic cells and metastatic colorectal cancer cells

Human metastatic colorectal carcinomas (CRCAs) express carcinoembryonic antigen (CEA) and/or MUC1 tumor-associated antigens as potential targets for the induction of active specific immunity. In the present study, freshly isolated metastatic CRCA cells were successfully fused with immature autologous human monocyte-derived dendritic cells (DCs). The created heterokaryons (DC/CRCA) coexpress the CRCA-derived CEA and MUC1 antigens and DC-derived MHC class II and costimulatory molecules. The fusion cells were functional in stimulating the proliferation of autologous T cells. In addition, both CD4(+) and CD8(+) T cells were activated by fusion cells, as demonstrated by the production of high levels of IFN-gamma. More importantly, coculture of fusion cells with patient-derived peripheral blood mononuclear cells (PBMCs) resulted in the induction of antigen-specific cytotoxic T lymphocytes (CTLs). CTLs were effective at lysis of not only autologous CRCA cells but also the CEA and/or MUC1-positive and HLA partially matched target cells. Antigen-specific CTL responses were confirmed by tetrameric analysis. Coculture of PBMCs with fusion cells resulted in increased frequency of CEA- and MUC1-specific CTLs simultaneously. Taken together, these results indicate that freshly isolated human metastatic CRCA cells expressing the CEA and/or MUC1 may represent a potential partner for the creation of DC/tumor fusion cells targeting induction of antigen-specific CTL responses. Our report demonstrates the simultaneous induction of CRCA-specific CTL responses restricted by HLA-A2 and -A24.

Heterogeneity within Antigen-Specific T Cell Responses Revealed by Differential Dynamics of TCR Downregulation.

Cytomegalovirus (CMV) is a known cause of significant morbidity, including CMV pneumonitis, among bone marrow transplant patients. Studies investigating the immune response to CMV pneumonitis within bronchoalveolar lavage samples illustrate a predominance of CD8 T cells. However, CD8 T cell infiltration does not correlate with clinical response. Likewise, cancer vaccine trials have failed to show a correlation between induction of antigen-specific CD8 T cell populations and clinical tumor response. Based on the recent finding that T cells downregulate the T cell receptor (TCR) upon activation, we demonstrate by stimulating antigen-specific T cells with graded amounts of cognate peptides that individual cells lose tetramer reactivity with different dynamics which reflects previously uncharacterized heterogeneity within the population. We analyzed response among virus-, CMV PP65 and EBV BMLF1, and tumor associated antigen- (TAA), MART and gp100, specific CD8 T cell clones and patient samples. Critical T cell response characteristics including sensitivity to antigen, recognition efficiency (RE, also referred to as functional avidity), and relative functional state were calculated based on TCR downregulation. We show TCR downregulation represents an accurate assessment of average T cell function with direct correlation to killing capacity by chromium release (r2 of.907, P<0.01) and degranulation by CD107 mobilization (r2 of.99, P=0.016). However, despite correlation of average T cell function, we found heterogeneity in T cell sensitivity and relative functional status not evident by chromium release or CD107 mobilization alone. TCR downregulation among viral clones with homogeneous tetramer+ populations appeared similar, however one CMV-specific clone was unique in lower RE (7.18, group mean 7.32) and poor functional status (29.6% of tetramer+ CD8 T cells failed to downregulate at supraphysiologic concentrations of stimulating peptide, group mean 9.1%) despite the highest sensitivity (11.6, group mean 8.9). Among CMV-specific patient samples, size of tetramer+ population was not correlated with CD8 T cell sensitivity or RE. Patient 10539 with the largest tetramer+ population (1.8%, group range 0.5–1.8%), demonstrated the lowest sensitivity (10.2, group range 10.2–12.3) and only average RE (9.2, group range 7.8–10.2). TAA-specific CD8 T cell clones and patient samples post-vaccination with heteroclitic peptide contained significant heterogeneity in response to native and heteroclitic peptides. Despite equivalent percent tetramer+ populations, gp100-specific patient samples were significantly less sensitive (8.9 vs 10.8) with lower RE (7.3 vs 9.2) and greater percent relatively nonfunctional (38% vs 2%) to native versus heteroclitic peptide. These findings suggest failure of CD8 T cells specific for CMV or tumor antigens to produce a clinically significant response may be due to heterogeneity in the T cell population. Specifically, in states of low antigen expression, such as chronic viral infection and cancer, CD8 T cells of high sensitivity and RE with adequate functional status may be required for competent in vivo immune response. It is thus important to understand and appreciate the heterogeneity which may exist within antigen-specific T cell populations, and its clinical implications, to better predict efficacy of the immune response to cancer vaccination or following bone marrow transplant.

Vaccines in breast cancer

<h2>SUMMARY</h2> Several antigens recognized by cellular and humoral effectors of the immune system have been identified in breast cancer. ‘Cancer/Testis' antigens, e.g. MAGE, NY-ESO-1, CT-7; differentiation antigens, e.g. NY-BR-1, CEA; overexpressed antigens, e.g. p53, HER2/neu, MUC-1, LewisX; and mutational antigens, e.g. p53, represent potential targets for antigen-specific immunotherapy. Clinical vaccine studies, mostly performed in melanoma, and aimed at the induction of antigen-specific CD8+ T cell responses in vivo, have helped to establish sensitive tools for the monitoring of immune responses to the vaccine in vivo and in vitro. Delayed-type hypersensitivity (DTH) reactions observed after intradermal injection of antigenic peptides were found to closely correlate with the induction of antigen-specific CD8+ T cell responses. Cytokines (GM-CSF, IL-2, IL-12) and adjuvants (QS21, IFA, MHC class II restricted helper peptides) have mediated increased CD8+ T cell responses against different antigenic peptides. The site of immunization may have important implications for the quality of immune responses induced. Intradermal and intralymphatic vaccination has led to strong CD8+ T cell responses in vivo, measurable at the immunization site, in metastatic lesions and in the peripheral blood. Intravenous and subcutaneous vaccination have induced measurable immune responses less frequently. Different strategies of vaccination using dendritic cells loaded with antigenic peptides, proteins or lysates prepared from autologous or allogeneic tumour cells have also led to measurable immune responses in vivo. Results, however, were not superior to vaccination with antigenic peptides alone or combined with adjuvants. Focusing on breast cancer, one of the most promising antigens for the design of vaccine studies is the CT-antigen NY-ESO-1, which was identified by the SEREX method. NY-ESO-1 is expressed in approximately 30–50% of breast cancers. Spontaneous humoral and cellular immune responses against NY-ESO-1 can be detected in 50% of patients with NY-ESO-1+ tumours. Specific DTH- and CD8+ T cell responses were induced after vaccination with NY-ESO-1 derived peptides alone and combined with GM-CSF in the majority of NY-ESO-1-naive patients. Measurable immune responses that can be correlated with the clinical outcome represent an important prerequisite for any immunotherapeutic intervention in cancer immunotherapy. At present, measurable but limited disease will teach us about any immediate effects of immunotherapy on cancer cells. This will lead the way in the near future to sound and justified adjuvant treatment strategies in the specific immunotherapy of breast cancer.

Induction of antigen specific CD4+ T cell responses by invariant chain based DNA vaccines

In this report, the use of DNA vaccination to induce class II restricted antigen specific proliferative responses was studied. To this end, a construct encoding the invariant chain (Ii) was engineered in which the Class II associated invariant chain peptide (CLIP) sequence was replaced by an immunogenic epitope derived form Heat Shock Protein 60, HSP60 178–186. Transfection studies in vitro showed that this construct can be used to efficiently load MHC class II molecules and present epitopes to MHC class II restricted antigen specific T cells. In addition, we showed that intradermal immunisation of Lewis rats with these constructs induced antigen specific T cells in vivo. Therefore, our Ii-gene constructs can be used to immunise for defined CD4+ T cell epitope sequences.

Induction of antigen-specific CD8+ T cells, T helper cells, and protective levels of antibody in humans by particle-mediated administration of a hepatitis B virus DNA vaccine

A DNA vaccine against the hepatitis B virus (HBV) was evaluated for safety and induction of immune responses in 12 healthy, hepatitis-naı̈ve human volunteers using the needle-free PowderJect™ system to deliver gold particles coated with DNA directly into cells of the skin. Three groups of four volunteers received three administrations of DNA encoding the surface antigen of HBV at one of the three dose levels (1, 2, or 4 μg). The vaccine was safe and well tolerated, causing only transient and mild to moderate responses at the site of administration. HBV-specific antibody and both CD4+ and CD8+ T cell responses were measured before and after each immunization. All the volunteers developed protective antibody responses of at least 10 mIU/ml. In volunteers who were positive for the HLA class I A2 allele, the vaccine also induced antigen-specific CD8+ T cells that bound HLA-A2/HBsAg 335–343 tetramers, secreted IFN-γ, and lysed target cells presenting a hepatitis B surface antigen (HBsAg) CTL epitope. Enumeration of HBsAg-specific T cells producing cytokine indicated preferential induction of a Type 1 T helper cell response. These results provide the first demonstration of a DNA vaccine inducing protective antibody titers and both humoral and cell-mediated immune responses in humans.