We focus on elucidating new fundamental principles governing T cell activation, regulation, and effector function, and employ these to develop more effective vaccine and immunotherapy strategies for human immunodeficiency virus (HIV) and cancer. This involves several steps that together comprise a push-pull approach. First, we optimize the antigen to improve immunogenicity by epitope enhancement, changing the amino acid sequence to increase affinity for the relevant major histocompatibility (MHC) molecule. This has been done for several antigens, including 2 new prostate cancer antigens, TARP and POTE, and we are completing a phase I/II clinical trial in D0 prostate cancer patients with rising PSA levels to determine whether the TARP peptide vaccine can reduce the rate of PSA rise. Some patients have had significantly increased PSA doubling times. We also enhanced a peptide from HIV reverse transcriptase that is mutated in response to lamivudine, and we are collaborating on a clinical trial with Robert Yarchoan (NCI) to determine whether this vaccine can provide selective pressure against outgrowth of drug-resistant mutant HIV. The second step is to push the immune response with molecular adjuvants (e.g., cytokines, Toll-like receptor (TLR) ligands) to improve not only its quantity but also its quality. We showed that IL-15 is an important mediator of CD4 T cell help for CD8 T cells, being sufficient to substitute for help in animals depleted of CD4 T cells, to allow a memory CD8 response and prevent TRAIL-mediated apoptosis, and is also necessary for help. If dendritic cells (DCs) cannot be induced by helper cells to make IL-15, then the help is not adequately delivered to CD8 T cells. Previously, we found that IL-15 increased the avidity of CD8 T cells, which are necessary for effective clearance of virus or tumor cells. We translated this to humans, showing that IL-15 could substitute for CD4 help to induce a primary in vitro CD8 T cell response of naive T cells, whereas IL-2 could not. IL-15 also restored the alloresponsiveness of CD4 T cells to normal levels in HIV-infected patients. We also investigated TLR ligands as adjuvants, because these can mature DCs and induce their production of cytokines like IL-12 and IL-15. We showed synergy between pairs of TLR ligands that work through different intracellular signal transducers, MyD88 or TRIF, and determined the mechanism in DCs involving unidirectional cross-talk from TRIF to enhance MyD88-dependent cytokine production. We showed that a triple TLR ligand combination induces more effective protection against virus infection, does not increase T cell quantity, and actually improves quality by inducing higher avidity T cells and more IL-15 production. We tested a combination of triple TLR ligands, IL-15, both or neither as adjuvants in a peptide-prime, MVA-boost mucosal vaccine for simian immunodeficiency virus (SIV) in macaques, challenging intrarectally with SIVmac251. Only the macaques receiving both adjuvants showed some protection, so we then investigated the correlates of this protection. Surprisingly, in the adaptive immune arm, only polyfunctional CD8 T cells specific for SIV antigens, but not total specific T cells as measured by peptide-MHC tetramer binding, correlated with protection. In the innate immune arm, the adjuvants induced long-lived innate protection by APOBEC3G. Thus, molecular adjuvant vaccines that induce both innate and adaptive immunity may be the most efficacious. The third step is to target the immune response to the relevant tissue;the mucosa in the case of HIV. We published a study of mucosal T cell trafficking in which we discovered a lack of equilibrium between T cells populating the intraepithelial compartment and the lamina propria in the small intestine, leading to a distinct founder effect causing a narrower repertoire of intraepithelial T cells. We also found that homing to the large intestine is governed in part by DCs from colon patches, using a mechanism that is distinct from that found in the small intestine. We are now trying to develop approaches to selectively target T cells to the large intestine. We also developed a novel approach to vaccine delivery to the large intestine, using nanoparticles coated with Eudragit FS30D to allow oral delivery and release of these particles primarily in the large intestine, bypassing the stomach and site of oral tolerance in the small intestine. This approach could replace intrarectal delivery to induce protection against viral challenge via the rectal or vaginal route. The fourth step is to pull the response by removing the brakes (i.e., block the negative regulatory mechanisms that inhibit immune response). We previously discovered a new immunoregulatory pathway involving natural killer T (NKT) cells that suppress tumor immunity. The NKT cells make IL-13 that induces myeloid cells to make TGF-beta, which suppresses the CD8 T cell response. However, NKT cells can also protect against tumors, so we needed to resolve this paradox. We found that type I NKT cells (using an invariant TCRalpha chain) protected, whereas type II NKT cells (using diverse T cell receptors) suppressed immunity. Moreover, selective activation of type I or type II NKT cells showed that these cells cross-regulated each other, defining a new immunoregulatory axis analogous to the axis between Th1 and Th2 cells that has profoundly affected immunology. The balance along the NKT axis could influence subsequent adaptive immune responses. We found that type II NKT cells also suppress conventional CD4 and CD8 antigen-specific T cells, so these cells are broadly suppressive. We are examining the mechanisms of suppression and investigating the relationship between suppressive NKT cells and CD25+ Foxp3+ T regulatory cells. Conversely, stimulating with a type I NKT cell agonist can protect against tumors. To expand on potential NKT agonists, we examined a beta-mannosyl analog of alpha-galactosylceramide, and found that, in contrast to other beta-linked sugar glycolipids, this one is also protective. However, its mechanism of protection against cancer is different from that of the classic alpha-GalCer, because it is dependent on TNF-alpha and nitric oxide synthase rather than on interferon-gamma and it synergizes with alpha-GalCer. This analog also stimulates human NKT cells, so it represents a new class of NKT cell agonists that could be translated to cancer therapy. TGF-beta is a key mediator of the NKT regulatory pathway and an important regulator of T regulatory cells. We found that blockade of TGF-beta can protect against certain tumors in mice, and can synergize with anti-cancer vaccines in two mouse models (published). The protection is dependent on CD8 T cells, and when used in combination with a vaccine, the anti-TGF-beta increases both the number of total and high avidity CD8 T cells. We translated this finding into a Phase I clinical trial of a human anti-TGF-beta monoclonal antibody (CRADA with Genzyme) in melanoma and renal cell cancer. This study showed some activity (1 long partial remission, 2 mixed responses, and 2 cases of stable disease among 22 patients). We are now developing phase II clinical trials in melanoma and prostate cancer, and a combination trial with a vaccine to induce anti-cancer immunity. Finally, we recently published that an adenovirus vaccine expressing the extracellular and transmembrane domains of HER-2 can cure large established mammary cancers and lung metastases in mice. Surprisingly, the mechanism involves antibodies that inhibit HER-2 function, rather than T cells. We are now making a similar cGMP recombinant adenovirus expressing the human HER-2 domains to carry out a clinical trial in cancer patients and have already confirmed expression of HER-2.