Immuno-gene therapy is a promising approach for the control of cancer. Because of the essential role in activating and expanding T cell responses to antigens, cytokines are comprehensively studied for cancer therapy. The efficacy of cancer immunotherapy is diminished by the fact that spontaneous tumors often downregulate antigen expression and/or presentation. The density of tumor antigen in conjunction with major histocompatibility complex (MHC) class I molecules on the cell surface affects cytotoxic T cell (CTL) function. In this study, we have developed a novel approach to augment the effect of cytokine-based cancer immuno-gene therapy through the coupling of antigen expression/presentation with cytokine production by expression of antigen epitopes within cytokine signal peptides. We first investigated the possibility of modifying cytokine signal peptides with antigen epitopes without aborting function. We inserted the genes encoding the MHC class I restricted antigenic epitopes of chicken ovalbumin (OVA), tyrosinase related protein 2 (TRP-2), and oncoprotein HER2 into the signal sequence of the interleukin-2 (IL-2) gene, replacing part of the signal sequence at different positions. Our results showed that these modified signal peptides still functioned, as indicated by cytokine secretion. We then demonstrated that an antigen epitope contained within the modified signal peptide could be processed properly and presented on the tumor cell surface. Furthermore, using a murine melanoma model, we studied the effect of antigen epitope modified IL-2 and IFN-gamma on the immuno-gene therapy of malignancy. Tumor cells showed enhanced immunogenicity as indicated by increased susceptibility to CTL lysis in vitro and decreased tumor growth in vivo after gene modification with the antigen epitope-containing cytokine expression vectors. Vaccination with TRP-2 epitope-containing IFN-gamma gene-modified B16 cells resulted in protection against wild type tumor challenge. In addition, we investigated the possibility of using antigen epitope-containing cytokine expression plasmids as DNA vaccines. Our data showed that immunization with an OVA epitope-modified IL-2 expression plasmid resulted in protective immune responses to an OVA expressing tumor. In summary, the work presented here demonstrated that enhanced immunological effects could be achieved through coupling cytokine expression with antigen presentation. These findings provide potential perspectives in developing therapeutic or prophylactic vaccines for immuno-gene therapy of cancer.

Recombinant DNA vectors are fundamental tools in gene therapy research. A novel cloning system, pLinus, was made to facilitate vector construction by providing 32 unique restriction sites to adapt DNA fragments in a single step. To compensate the low delivery efficiency of the non-viral vector systems, we have constructed two high expression plasmid vectors, pHi1/2, by incorporating a transcriptional amplifier strategy into a single construct. In both pHi1/2 vectors, the amplifier expression cassettes contained two independent transcriptional units. One transcriptional unit contained a transcriptional factor, the tat gene, driven by a strong constitutive CMV promoter. The second transcriptional unit contained either an HIV1 LTR or HIV2 LTR driving the gene of interest. Using the human IL-2 cytokine as a reporter and therapeutic gene, the pHi1/2 amplifier vectors could achieve significantly higher IL-2 expression levels than that observed when using the CMV promoter alone. In vivo injection of the stable pHi2-IL-2 gene modified Lewis Lung (LL/2) tumor clones resulted in slower tumor growth and longer survival as compared to those mice injected with either CMV-driven IL-2 transfected clones or the parental tumor cells. To solve the safety concern, we constructed a novel plasmid vector, pHi-Hot, by combining inducible and amplifier strategies in a single vector. In pHi-Hot, the first transcriptional unit contained an inducible heat shock protein (hsp70B) promoter controlling the expression of a transcriptional factor, Tat, which transactivates a second promoter, the HIV2 LTR, located downstream on the same construct. The second promoter drives the gene of interest. Using the human IL-2 cytokine gene as a reporter gene, we demonstrated that, heat shock at 42°C for 30 min, the pHi-Hot vector could achieve high gene expression levels while maintaining its inducibility. The induced IL-2 levels were significantly higher than achieved by using the hsp promoter or CMV promoter directly. And repeated heat shock at 42°C for 30 min of mice injected with a pHi-Hot-IL-2 gene modified LL/2 clone led to tumor regression. In this study, three major approaches towards facilitating vector construction and improving vector expression cassette design are described.

RSV infections are ubiquitous in children but are most severe in infants in the first few months of life. Innate and adaptive immune responses were assessed to respiratory syncytial virus (RSV) in neonate mononuclear cells (MCs) obtained at birth (prior to any direct and independent exposure) and these responses were compared to those from adult MCs. Our hypothesis was that neonates exhibit innate and/or adaptive immune hyperresponsiveness to RSV. In neonate MCs, inactivated virus invoked large levels of the innate immune cytokines IL-6, TNF-α, and IL-10 and low levels of IFN-γ and IL-12 but no adaptive immune cytokines. Live RSV induced lower levels (compared to inactivated virus) and fewer innate (IL-6, IL-10 and IFN-γ) and no adaptive immune cytokines. In adult MCs, inactivated and live virus invoked cytokines reflecting both innate and adaptive immunity (IL-6, IFN-γ, IL-2, TNF-α and IL-10). Further, NK cells (and not T cells) were the primary source of IFN-γ in neonate MCs, whereas both NK cells and T cells contributed towards IFN-γ in adult MCs to live RSV. RSV-induced proliferation in neonate MCs was not observed, though positive in adult MCs. Taken together, the results indicated that the immune response to live virus at birth was only innate in nature and could be characterized as hyporesponsive (compared to adults), thus contradicting our initial hypothesis. Immune suppression to live RSV in neonate MCs was found to be closely associated with dysregulation between type I (IFN-α) and type II (IFN-γ) IFNs. IFN-α expression in neonate MCs was significantly more than in adult MCs to live RSV. Inhibition of the live RSV-induced IFN-α in neonate MCs led to significant increases in innate (IFN-γ, IL-12, IL-18 and TNF-α) immune cytokine production. Interferon regulatory factor-1 (IRF-1) expression was part of the immune response to live RSV in adult MCs though not in neonate MCs and that the lack of IRF-1 upregulation in neonate MCs was reversed when IFN-α was neutralized. Overall, enhanced IFN-α expression suppressing innate immune cytokine production and IRF-1 expression in neonate MCs may account for the severity of early life RSV-induced illnesses.

In this dissertation we investigate the role of avidity in the T cell selection process by examining the impact of signal modulation on T cell and/or NKT cell development. Projects discussed herein (including peptide, anti-CD1d, and hydrocortisone (HC) therapy) examine how changes in avidity can be used to explore potential therapies for Type 1 diabetes mellitus (T1DM). In the case of peptide therapy, we find that fetal thymic organ culture (FTOC), treated with exogenous diabetes related GAD peptides, lose their ability to generate T cell responses to GAD treatment peptides. Also, peptide therapy is shown to inhibit T1DM in vitro (ivT1DM) and in vivo. The abnormally high level of GAD peptides that are presented during peptide therapy treatment are thought to increase avidity between peptide specific T cells and selecting cells during thymic education, leading to increased negative selection of those T cells. In the case of anti-CD1d, FTOC from C57BL/6 (B6) and non-obese diabetic (NOD) mice, when treated with 10 μg/mL of anti-CD1d, show divergent responses to treatment. In response to anti-CD1d, "normal" B6 FTOC shows decreased T cell development and NKT production. Conversely, "poor signaling" NOD mice show no major impact on general T cell development but instead show increases in NKT cell production. Also, treatment with anti-CD1d is shown to inhibit diabetes in our ivT1DM model. These effects are thought to be due to increases in avidity generated through anti-CD1d related increased TCR expression. Changes in avidity caused by anti-CD1d treatment are thought to generate increased negative selection in B6 FTOC, while the same avidity increases are thought to increase positive selection (without increasing negative selection) in "poor signaling" NOD FTOC. In the case of HC treatment, B6 FTOC treated with HC show changes in T cell yield, maturity, and TCR Vβ usage. Research with HC indicates that signal inhibitors have the capacity to change T cell development in a dose and time dependent manner. Based on this work, selection signal inhibitors or enhancers may have the capacity to change T cell development in a fashion that decreases autoimmune T cells and/or enhances regulatory NKT cell development.

This work introduces and explores a novel model which incorporates Fetal Thymus Organ Culture (FTOC) from non-obese Diabetic (NOD) mice to replicate thymic development and function of diabetogenic T cells in vitro. NOD FTOC is shown to posses a predictable diabetogenic activity measured in vitro, and a protective, regulatory activity when adoptively transferred to genetically IDDM-predisposed NOD mice. This in vitro IDDM (ivIDDM) activity is unique to NOD FTOC, and can be abbrogated by co-culture of developing NOD FTOC with FT from immunologically incompetent C.B-17 scid/scid mice. Additionally early exposure of NOD T cell precursors to islet antigens by co-culture with NOD Fetal Pancreas can negatively select for diabetogenic T cells or activate immuno-regulatory cells that can suppress diabetogenic T cell activity. The addition of blocking F(ab' )2 fragments of anti-CD3epsilon monoclonal antibody to NOD FTOC/FP co-cultures prevented insulin reduction, implicating a role for TcR-mediated recognition in this "in vitro IDDM" model. Transfer of unprimed syngeneic FTOC cells to pre-diabetic NOD mice prevents the onset of IDDM while transfer of islet-cell primed FTOC/FP cells slightly increased disease incidence. Spontaneous proliferation to peptides of Glutamate Decarboxylase (GAD) was not detected in NOD FTOC in contrast to reports of such responses in pre-diabetic NOD mice. A marked response to GAD peptides is induced by priming NOD FTOC, and increases ivIDDM. Proliferation is significantly diminished by tolergenic early treatment of FTOC, as is ivIDDM activity. Offspring of GAD peptide-treated NOD mice have a lower incidence of IDDM indicating potential beneficial tolerance to islet antigens by in utero exposure. Injection of identical GAD peptides to pre-diabetic NOD mice enhances the incidence of IDDM demonstrating the deleterious effects of inappropriate in vivo administration of autoantigenic peptides. NOD FTOC is shown to readily integrate and express retrovirus-delivered class II MHC I-E(α)d as measured by PCR and flow cytometry, respectively. The contribution to understanding the antigenic, genetic and regulatory basis of IDDM in NOD mice and humans is discussed.

T cells recognize antigenic peptides presented by MHC molecules on antigen presenting cells (APC) through T cell receptors (TCRs). Since TCRs are very similar to antibodies in structure and genetics, TCRs might have the potential to bind free antigens as antibodies do. Here, peptides which bound TCRs irrespective of MHC molecules have been identified by screening "one-bead one-peptide" combinatorial libraries. Peptides: VRENAR, RTGNYV, GKMHFK, KDAVKR and RKPQAI bound recombinant Jurkat single chain T cell receptors (scTcrs). GKMHFK, KDAVKR and RKPQAI were also specific for natural TCRs on the Jurkat cell surface. Molecular modeling implies that Glu96 in the CDR3 loop of TCR alpha chain is a candidate for the peptide interaction site. However, TCR-binding peptides did not induce biological effects on parental Jurkat cells. To extend this study to a biologically relevant system, diabetogenic T cells involved in insulin-dependent diabetes mellitus (IDDM) have been characterized. GAD(524-543) responding T cells showed restricted TCR variable gene usage, which utilized preferentially Vα17 and Vβ12. Three domain single chain T cell receptors (3D scTcr) were constructed as tools to investigate potential therapies for IDDM and to identify peptides which bind to TCR without association of MHC molecules. Functional analysis has demonstrated that GAD(524-543)-specific scTcrs retained the ability to bind GAD(524-543)/IAg7 complex. This work shows that recombinant scTcrs can bind cognate peptide presented by MHC molecules, therefore they can be used as substitutes for natural TCRs in screening "one-bead one-peptide" combinatorial libraries to identify TCR-binding peptide.

IFN-γ and IL-4 are cytokines that are thought to be products of distinct differentiation pathways for lymphocytes and that inhibit and enhance the development of IgE antibodies, respectively. We hypothesized that the level of IFN-γ production upon stimulation of immune cells in the blood is a stable, heritable phenotype that is inversely related to phenotypic markers of allergy and to the Th2 cytokine IL-4. The studies were performed with samples of human blood obtained from children enrolled at birth in a large, longitudinal study of allergy known as the Tucson CRS. Mitogen-stimulated IFN-γ production by PBMCs of children at age 11 was compared to IFN-γ production in the first year of life and found to show a significant positive correlation. The expected inverse relation to markers of allergy (total serum IgE, skin test reactivity and eosinophils) was not observed. A strong positive relation was observed between IFN-γ production in the parents and children, lending support to the concept of genetic regulation. In contrast to the inverse relation hypothesized to IL-4, a positive relation was observed to IL-4 as well as to another cytokine, IL-2. A linkage analysis performed by a colleague showed linkage between low production of IFN-γ and a region of chromosome 5q in which the gene for a T-cell specific kinase (Itk) is located. These results led us to hypothesize a common "regulator" of cytokines that was functioning and might be Itk. Decreased expression and/or decreased function of Itk could account for the low cytokine data. In order to test this hypothesis, ten adult volunteers with low and ten with high cytokine production matched for age and sex were tested for Itk function and expression. The two groups were examined for Itk expression (normalized to CD3 expression) by western blotting. The group of low cytokine producing individuals showed significantly less Itk expression than the high cytokine-producing group. Attempts to optimize an in vitro kinase assay to determine Itk function were unsuccessful. In the absence of functional studies to support the results from the expression studies, it is not possible to say with certainty that Itk function is related to cytokine production. Therefore, we conclude that IFN-γ expression in humans is a stable phenotype with evidence of a genetic basis for regulation and that Itk is a candidate gene contributing to that genetic basis.

Natural autoantibodies specific for the T cell receptor (TCR) are present in all human sera. Differences in titer, epitope specificity, and isotype depend on physiological condition, viral infections, or the presence of autoimmune diseases. Individuals with rheumatoid arthritis (RA) make significantly higher titers of IgM isotype autoantibodies demonstrating major reactivity for the CDR1 region of the Vβ TCR. To establish a more intimate understanding of the role of these antibodies in the immune system we generated B cell hetero-hybridomas secreting monoclonal IgM autoantibodies from the synovial tissue and peripheral blood of RA patients. We report molecular and partial functional characterization of seven IgM anti-TCR monoclonal antibodies (mAbs). These autoantibodies were selected on a recombinant TCR and peptide epitopes and bind JURKAT human T cell lines and a subset of CD3⁺ human peripheral blood mononuclear cells (PBMCs) in flow cytometry experiments. The V regions of these antibodies were generally identical to germline sequences in both the heavy and the light chains and the heavy-chain CDR3 segments did not correspond to known antibody sequence. Three of these anti-TCR mAbs, OR2, ORS, and Syn 2H-11, demonstrated an antigen specific binding property defined as epitope recognition promiscuity. The molecules did not act as rheumatoid factors. These same mAbs bound to subsets of murine T cells and TCR peptide epitopes. The autoantibodies did not induce apoptosis in vitro, but prevented IL-2 production by antigen-specific T cells. These findings describe a unique group of immunoregulatory antibodies and represent a foundation for further investigations and their eventual use as therapeutic agents in human disease.

It is well documented that long-term spaceflight adversely affects immune system function. Using fetal thymus organ culture (FTOC), we examined the effects of spaceflight and vector-averaged gravity on T cell development. In order to perform this work, we needed to design and validate a culture system that supported FTOC in a microgravity environment. The system we built, and which is described herein, served successfully for ground-based experimentation built on the principle of the clinostat. Moreover, results of testing this system at NASA's Ames Research Center demonstrated that it is optimal for future flight experimentation. This system can also be used for other cell/organ culture methodologies where three-dimensional growth and organotypic organization optimizes function for ground-based as well as spaceflight experiments. Under both conditions (spaceflight and vector-averaged gravity), the development of T cells was significantly attenuated. Exposure to spaceflight for 16 days resulted in a loss of precursors for CD4⁺, CD8⁺, and CD4⁺CD8⁺ T cells in a rat/mouse xenogeneic co-culture. A significant decrease in the same precursor cells, as well as a decrease in CD4⁻ CD8⁻ T cell precursors, was also observed in a murine C57BL/6 FTOC after rotation in a clinostat. The observed block in T cell development appeared to occur between the pre-T cell and CD4⁺CD8⁺ T cell stage. Furthermore, flow cytometric analysis clearly illustrated a reduction in the expression of IL-7Rα (CD127) in clinorotated FTOC as well as an increase in the presence of TNF-α after 4 days of culture. Levels of phosphorylated Lck were unchanged in clinorotated FTOC when compared to motional and stationary controls. These findings suggest that the full sequelae of pre-TCR signaling is dependent upon the presence of gravity. However, this alteration may be occurring downstream of Csk/Lck regulation. In support of this line of reasoning, T cell development was partially rescued in clinorotated FTOC treated with anti-CD3 monoclonal antibody. Anti-CD3 treatment presumably partially substitutes for a signal that is required for proper T cell development and is absent in the microgravity environment. These data therefore indicate that gravity indeed plays a decisive role in β-selection and in broader terms the development of T cells.

Although several commercial serological kits exist for Toxoplasma serodiagnosis, the unambiguous diagnosis of many clinically important Toxoplasma infections remains problematic. This is particularly true in establishing the timing of infection in pregnant women and in demonstrating reactivation of disease in immunocompromised hosts. The wide tissue tropism and distinct life-cycle stages of toxoplasmosis raise the possibility that the detection of circulating tachyzoite antigens may be of use in these situations. We have developed a series of antigen capture Enzyme Linked Immunosorbent Assays (ELISAs) using a panel of novel monoclonal antibodies (mAbs) directed against T. gondii tachyzoite antigens. Using a pool of these mAbs to capture whole tachyzoite lysate antigen in 'spiked' negative serum, the detection limit of our ELISA was 1--2mug/ml of protein. The sensitivity of this ELISA was 52% (n = 412). We postulated that our low sensitivity was mainly due to circulating immune complexes. This was confirmed by the disappearance of 'spiked' tachyzoite lysate antigen in antibody positive samples followed by the reappearance of antigen upon 12% trichloroacetic acid (TCA) treatment. Furthermore, the use of 12% TCA significantly increased antigen detection (p = 0.00001). Preliminary results suggested that assay sensitivity was 96% (n = 254) while assay specificity was 97% (n = 253). Early reports suggest that Toxoplasma antigen levels in serum are transient. The magnitude and kinetics of antigenemia with the specific Toxoplasma products recognized by our panel of mAbs remain to be determined. This optimized assay can now be used to test sera from otherwise healthy and immunocompromised subjects to determine its clinical utility.