An immune response can be specific both in effector function and localization. T-cells play a critical role in the control and evolution of an immune response. T-cells generally recognize small peptide fragments of protein antigens that are bound to major histocompatibility complex (MHC) molecules. The T-cell receptor (TCR) and the signaling cascade associated with TCR ligation can discriminate between similar antigen fragments bound to MHC molecules. These small antigenic differences can alter the T-cell response intensity or phenotype or even antagonize responses to other antigens. In addition, co-receptors that recognize conserved areas on MHC molecules, receptors for cell-surface adhesion/co-stimulatory molecules, and receptors for soluble mediators can all modulate the outcome of T-cell activation. These inputs depend on the structure and processing of the antigen, the cell surface molecules on the antigen-presenting-cell (APC) and neighboring cells, and the secretion of soluble mediators such as chemokines and cytokines into the local microenvironment. These varied inputs are integrated and lead to specific T-cell outputs through a complex kinetic reorganization of molecules at the cell surface of T-cells and multiple interlacing signal transduction pathways. The outcome of the T-cell activation process leads to specific changes in the T-cell phenotype that control migration, survival, proliferation and effector functions. After migration of the activated T-cells to their next destination, these characteristics, combined with the responses of the other immune cells at the same location and the local microenvironment, are incorporated into the overall immune response. To isolate the effects of specific adhesion/co-stimulatory molecules and their combinations, we had previously developed an antigen-presenting-cell (APC)-free system for stimulation of naive T cells. In this system naive T-cells from T-cell-receptor (TCR) transgenic mice were activated with antigenic peptide-MHC complexes bound to plastic plates. We demonstrated that potent antigenic peptide-MHC complexes can activate CD8+ T cells, leading to IL2 production and acquisition of lytic effector function. Different peptide stimuli led to a less intense T-cell response. The ability to vary the peptide-MHC potency is an important advantage of this system. Using this system we demonstrated that purified ICAM-1 molecules can further increase the proliferative and cytokine responses of naive T cells to purified MHC-peptide complexes. This enhancement was seen in the absence of the defined geometric pattern of molecules or organized immunologic synapse seen with different experimental systems. We are interested in evaluating other adhesion/co-stimulatory molecules, individually and in combination, using this APC-free system. Although our APC-free system has demonstrated that co-stimulatory molecules and the organization of an immunologic synapse are not absolute requirements for CD8+ T-cell activation, it cannot assess the complex dynamics between T-cells and APCs, or T-cells and microenvironments. Our lab has developed expertise in the cell-surface organization of T-cells and in evaluation of in vivo immune responses. We plan to assess the effects of varying TCR ligands and varying co-stimulatory/adhesion molecules using transgenic TCR T-cells across three model systems: a reductionist system with purified components and basic readouts, a microscopic readout of spatial changes in cell surface molecular organization and an in vivo system which can assess tissue specific effects with immunohistochemistry. The use of these systems with similar TCR stimuli will allow some integration of a detailed reductionist view of immunology by evaluating molecular, cellular, and tissue views of similar T-cell antigen interactions. The integration of the vast amount of data on factors that modulate an immune response at the molecular, cellular, and tissue levels is a difficult but critical task. We plan to look for differences between in vitro and in vivo responses and then evaluate potential mechanisms at the molecular, cellular, and tissue levels. This and related work can help bridge the gap between a myriad of individual molecular interactions often characterized by pleiotropy and redundancy and the outcome of in vivo challenges to the immune system. Activation and Modulation of Naive T Cells. Information regarding the minimal requirements for activation of primed T cells is readily available. However, the immune system is most amenable to modulation before completion of antigen priming. This project is to define the requirements for activation of unprimed naive T cells and to evaluate the effects of additional stimuli on the magnitude and type of T cell activation elicited. With the use of a potent antigenic peptide-MHC complex, we have been successful in the activation of phenotypically naive CD8+ transgenic T-cells. These T-cells have been stimulated to proliferate, secrete IL-2 and mature to cytolytic effectors in the absence of additional signals from antigen presenting cells. Through the use of this experimental system and purified recombinant soluble ICAM-1, we have evaluated the effects on T-cell activation of adding an isolated costimulatory signal. ICAM-1 can increase proliferation and cytokine production of CD8+ T-cells with under varying levels TCR signal intensity. We have also demonstrated that ICAM-1 has a differential effect on CD4+ and CD8+ T-cells and that the ICAM-1 enhancement of CD8+ T-cell activation can occur in the absence of an organized immunological synapse. Evaluation of other purified adhesion/costimulatory molecules may reveal different effects. To expand our readout of T-cell activation, we have added methodologies that have greater relevance to the in vivo outcome of T-cell activation. The use of transgenic T-cells in an adoptive transfer model has demonstrated the criticality of T-cell localization in the type of T-cell activation observed. This suggests the tissue distribution of immunomodulatory agents may have unexpected consequences. We have also evaluated microscopic changes in T-cells by studying the movement of the cell-surface mucin molecule CD43 after T-cell activation. These experiments have allowed us to assess subtle changes in T-cell activation. Using this system, we have been able to demonstrate immunologic effects of a statin HMG CoA reductase inhibitor. Although immunologic mechanisms have been suggested as a reason for the unexplained benefits of statins, our assay system demonstrates a specific microscopic readout for a statin effect on T-cells. This type of assay may facilitate comparisons of different statins for immunomodulatory effects. We are also planning to evaluate cytokine production and activation using primary human T-cells purified from buffy coats provided by the NIH blood bank. This enables extension of our results to naive human T-cells. This system will allow us to evaluate specific changes in naive T cell responses caused by the deletion or addition of lymphokines, adhesion molecules or costimulatory molecules. This can be done in the absence of unknown contributions from antigen presenting cells and correlated with cellular morphology and in vivo data using the same transgenic T-cells used in vitro. These results can be correlated with biochemical data on the involved molecules such as binding constants and thermodynamics by using SPR technology. We have performed this type of biochemical analysis on domains of the antigen presenting MHC1 molecule. Results from these studies have been used to develop mutant beta-2-microglobulin molecules that can interfere with peptide antigen presentation. The molecular requirements for T-cell activation and the varying phenotypes of this activation are critical for the generation of successful immunity and the modulation of aberrant immune responses. Understanding T-cell activation and how it can be modulated is important in assessing the mechanism of action of therapeutic antibodies directed against T-cells. Visualizing the Immune Response to Anthrax Toxins. There is limited data on the nature of the immune response that leads to protection from Anthrax. Antibodies to protective antigen (PA), a critical component of anthrax lethal and oedema toxins, have been shown to correlate with protection. However, studies suggest that other factors may play a role and little has been done to explore the role of T-lymphocytes in protection from and/or exacerbation of Anthrax. This lack of information regarding the localization and phenotype of T-lymphocyte responses to Anthrax toxins prevents design of appropriate therapeutics and improved vaccines. Development of immune based therapies and acellular Anthrax vaccines will require an understanding of Anthrax toxin antigen presentation and the response of T-lymphocytes to toxin antigen. We plan to address these questions using recombinant Anthrax toxins. These molecules will allow evaluation of toxin trafficking and antigen presentation of toxin. After generating data on antigen presentation with in vitro experiments using cell lines, the same recombinant toxins can be used for in vivo experiments. Histochemical evaluation of tissues from these animals will facilitate visualization of in vivo anthrax toxin localization and the subsequent immune responses. Visualizing the sub-cellular localization of Anthrax toxins and the in vivo immune response to these toxins can facilitate the design of assays to evaluate protein therapeutic effects on anthrax toxin. This information is important for development of safe and effective therapeutics and toxin subunit vaccines. Biosensor Sensitivity. Biosensors using specific plasmon resonance (SPR) are being used by industry for assessing biochemical parameters of protein therapeutics and immunogenicity. We are working to develop more sensitive methodologies for use of this technology. This could enhance detection of immunogenicity and allow use of this technology for sensitive detection of residuals in manufacturing. Clearance studies are often restricted by low assay sensitivity. Development of more sensitive assays could facilitate demonstration of increased clearance of manufacturing contaminants.
