The ability to generate potent T cell-mediated responses against self antigens is a prerequisite for the validation of cancer vaccines in humans. While a number of potential tumor-associated antigens (TAA) have been reported in literature, the clinical applicability of many of these antigens are often not properly followed with specific immunological studies. Advances in TAA-based immunotherapy have been hampered by absence of reliable animal models and inefficient vaccine approaches, in particular, poor adjuvants and delivery systems. TAA-induced immune responses often are suppressed by T regulatory cells (Tregs), a key cell type that enables evasion from anti-tumor responses. [unreadable] [unreadable] An ideal vaccine carrier should deliver the target antigen to antigen presenting cells (APCs), while, at the same time, promoting cellular activation. In this respect, the chemoattractant peptides appear to fulfill these requirements, in that they can both target and attract APCs, and in some cases (e.g., murine beta-defensin 2) can also activate cells via TLR-4. We have reported that non-immunogenic antigens were rendered immunogenic by fusing them to specific chemokine ligands to target them to APCs via chemokine receptors. In vivo studies have suggested that, in order to be effective, vaccines require the targeting of immature, but not mature dendritic cells (DCs). Moreover, the breadth of the vaccine strategy we have developed lies in the ability of chemokines to deliver TAA to the MHC class I antigen processing pathway. We have demonstrated that antigens delivered to chemokine receptors are efficiently internalized, processed to be subsequently presented to the MHC class I and II molecules. The chemokine-fused antigens were carried to early-late endosomal and lysosomal compartments through a clathrin-dependent process and subsequently processed by proteasomes in the cytosol, thus facilitating efficient cross-presentation through TAP-1-dependent processes. Typically, approaches that target various endocytic cell surface receptors are known to increase the efficiency of antigen presentation by 100- to 10,000-fold. In this respect, chemokine receptor targeting is also quite efficient in that only nmol (ng/ml) quantities of tumor antigens fused with chemokine are required to mount an effective response (Schiavo et al., 2006). [unreadable] [unreadable] One specific antigen we have focused our efforts on is OFA-iLRP, a highly conserved the 37-kDa oncofetal immature laminin receptor, is an embryonic antigen that is specifically and highly expressed in a number of human malignancies. Our studies have demonstrated that DNA immunizations with constructs expressing chemokines fused with OFA-iLRP elicit potent CD8+ T cell-mediated anti-tumor responses against syngeneic mouse tumors expressing this antigen, supporting the application of this vaccine as a possible cancer vaccine formulation for human use (Biragyn et al., 2007). Based on these data, we have initiated studies to test this vaccine in humans with non-small cell lung carcinoma (NSCLC).[unreadable] [unreadable] In addition, we have recently demonstrated that a novel sperm-derived antigen, SPANX-B, is specifically expressed in melanomas and lung cancer. Our results indicate that SPANX-B has significant clinical relevance, as SPANX-B is readily recognizable by T cells from healthy humans. At present, T cell epitopes of SPANX-B are being mapped and specific vaccine formulations are being generated. [unreadable] [unreadable] The recruitment and migration of cells, more specifically immune and malignant cells, are controlled by the network of chemokines and their cell-surface receptors. We have hypothesized that both immune responses and tumor growth may actually be controlled via delivering toxins through chemokine receptors. To test this, we have utilized the human T-lymphoblastoid CCRF-CEM cells (CEM) to imitate most human T cell lymphoma and adult T-cell leukemia lymphoma (ATLL) that express the chemokine receptor CCR4. We have produced novel chimeric proteins designated chemotoxin (chemokine linked to toxin), and demonstrated that chemotoxins targeting CCR4 were capable of efficiently killing CEM cells in vitro and in vivo in NOD-SCID mice with established CEM tumors. We believe that chemotoxins may have potential application when used alone or in combination with other therapeutic modalities to eradicate human T cell malignant diseases in immunocompromised patients (Baatar et al., 2007a).[unreadable] [unreadable] T-regulatory cells (Tregs) are thought to participate in tumor survival and suppression of vaccine-induced responses. To date, the precise nature of Tregs and the existence of different subsets of Tregs remain largely unknown. Characterization of Tregs has been complicated by the lack of unique markers as many Treg markers are also expressed on other immune cell types including activated T cells. Tregs migration, particularly skin-homing, is known to be controlled by chemokines. Using chemotoxins that specifically target chemokine receptors, we were able to demonstrate that non-fractionated Tregs actually consist of two distinct subsets, CCR4+ Tregs and CCR4- Tregs. These cells differ not only by their cell surface phenotype, but also by their functional properties. While CCR4+ Tregs appear to constitutively exert suppressive activity, CCR4- Tregs (possibly as adaptive or naive natural Tregs) require TCR-mediated activation to become fully active Tregs. Moreover, we have also demonstrated that Tregs utilized Fas-FasL (CD95-CD95L) signaling to mediate their immunosuppressive effects. This is not only an important finding on the Tregs biology, but also it opens possibilities to manipulate Tregs to combate cancer and autoimmune diseases (Baatar et al., 2007b). [unreadable] [unreadable] An important part of our research is also dedicated to understand immune responses to develop more efficient and safer immunotherapeutics. We have found that viral chemokines can serve as carriers to circumvent vaccine-induced anti-host chemokine immune responses. Moreover, we examined a novel and as-of-yet uncharacterized function of antimicrobial peptides, such as beta-defensin-like hypothetical peptide EP2c and demonstrated that EP2c can also be effectively utilized for targeting APCs. We believe that potency of carriers can be significantly improved if they directly promote the cellular activation and maturation of APCs. In this respect, we are continuing our previous study on the use of Toll-4 receptor by murine antimicrobial peptide, beta-defensin 2 (MBD2), to demonstrate that it also activates non-classical and membrane TNFR2-dependent cell death. [unreadable] [unreadable] Overall, we have developed and tested several strategies to manipulate chemokines to control immune responses and suppress cancer growth and metastasis. The use of chemoattractants as vaccine carriers is an efficient and simple strategy to elicit antitumor and vaccine responses. Taking advantage of chemokine redundancy, we have also demonstrated that viral chemokine fusions were equally potent in inducing protective immunity in vivo, providing a possible strategy to circumvent hypothetical, vaccine-induced anti-host chemokine autoimmunity. Chemotoxins can efficiently deplete unwanted subsets of cells. This strategy not only has clinical value for eradication of CCR4-expressing tumors, but also assists in examining the biology of Tregs.