In the previous (first) period of funding of this project we demonstrated that memory and effector CD8+ T cells exert reciprocal, DC-killing (""""""""suppressive"""""""") versus DC-activating (""""""""helper"""""""") activities and differentially affect the immunologic and anti-tumor activity of cancer vaccines. Our data demonstrate that effector CD8+ T cells kill antigen (Ag)-bearing DCs in a perforin (Pfn)- and Granzyme B (GrB)-dependent mechanism. In contrast, memory CD8+ T cells play a """"""""helper"""""""" role, inducing the DC expression of the serpin PI9 (CAP3/B9/SPI6), an endogenous GrB inhibitor, and protecting DCs from cytotoxic T cell (CTL)-mediated killing. They also induce type-1 polarization of DCs, manifested by their enhanced production of IL-12p70, enhanced ability to support Th1- and CTL responses and to mediate antitumor effects. Moreover, we have demonstrated that the nominally """"""""suppressive"""""""" effector CTLs can be converted to """"""""helper cells"""""""" following pharmacologic blockade of their cytolytic machinery, or following their TCR-independent activation with IFN? plus IL-18. Based on these data, we hypothesize that the elimination of tumor-associated antigenous GrB inhibitor, and protecting DCs from cytotoxic T cell (CTL)-mediated killing. They also induce type-1 polarization of DCs, manifested by their enhanced production of IL-12p70, enhanced ae propose to test the above hypotheses and to develop means to counteract the suppressive impact of pre-existing tumor-specific CD8+ T cells and to utilize their """"""""helper"""""""" potential in the following Specific Aims: 1. Identify the molecular mechanisms of DC-killing and DC protection/polarization by human CD8+ effector (Teff) versus memory (Tmem) cells, as potential targets of immunointervention. 2. Validate the key mechanisms of DC modulation by mouse CD8+ Teff and Tmem cells in vitro. Success of tehse studies will allow us to optimally design prospective in vivo mouse studies testing the relative contribution of the individual regulatory mechanisms to the Teff -and Tmem-mediated immune regulation in vivo and to develop strategies to counteract the CTL-mediated DC killing elimination and to utilize the CD8+ T cell-dependent help in mouse models of therapeutic cancer vaccination. The positive outcome of this project and it follow-up studies, will help us to understand basic principles of immune memory and regulatory functions of Teff and Tmem cells, and will allow us to develop new off-the-shelf therapeutic cancer vaccines and combined cancer therapies utilizing the principles of protection and polarization of endogenous DCs of cancer patients, in order to achieve continued immunologic and therapeutic effects of vaccination against established cancer.
Our current clinical trials that demonstrated early therapeutic promise of type-1-polarized DCs in patients with advanced melanoma, CTCL and glioma (see Progress Report/Preliminary Data) involve ex-vivo-manipulated DC, custom-generated from the blood of each individual patient. This type of therapy requires for highlyspecialized cGMP cell production facilities and is labor-intensive, resulting in relatively high costs of treatment (current costs of DC-based vaccines are within the range of $15-30K per patient, being high, but substantially less-expensive than most of the antibody-based cancer therapeutics), limit the possibility of its mass application. The possibility to develop DCs with more pronounced immunostimulatory function would help to amend this situation, by allowing the use of lower DC doses and potentially lesser numbers of immunization doses, reducing the scale of DC production and the costs. An even more radical solution towards large-scale implementation of immunotherapies based on the principle of DC1 polarization would be to develop a means of polarizing the patients'endogenous DCs in vivo, without their ex vivo processing. Our preliminary data indicate that both of these goals may be achievable by using CD8+ The studies perform within the next 2 years of funding will involve mechanistic studies aimed at definition of the basic pathways of DC modulation by effector and memory CD8 T cells to target endogenous DC, allowing us to develop type-1-polarized DCs in vivo, by promoting their interaction with memory-type CD8+ T cells. + Such prospective studies will allow us to T cells in human and mouse in vitro models. These of the identified mechanisms that operate in a similar fashion both in human and mouse systems will be prioritized for the (prospective) evaluation in mouse in vivo models of the immunologic and therapeutic activity as cancer vaccines. develop off-the-shelf vaccines In addition to these translational aspects, our studies will allow us to explain the long standing able to selectively boost type-1 immune responses and enhance therapeutic effectiveness of vaccination. We anticipate that the new therapies resulting from such (currently-proposed and prospective) studies will allow us to develop highly-feasible (no need for ex-vivo culture of DCs in cGMP conditions) life-prolonging treatments for cancer patients. controversy regarding the paradoxical ability of CD8+ T cells to both suppress and to promote immune responses observed in different experimental models, contributing to our overall understanding of the mechanism of immune memory.
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