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Days post HSV-2 infection

Figure 6 Protection from lethal HSV-2 challenge. Each group of mice (n = 10) was immunized with gD DNA vaccines (60 ^g), and/or cytokine genes (40 ^g) at 0 and 2 weeks. Three weeks after the second immunization, mice (n = 8) were challenged intravenously with 200 X LD50 of HSV-2 strain 186 (7 X 105 pfu).

observed to provide potent immune signals (138,139). They bind to their receptors (CD28/CTLA-4) present on T cells. The CD80 and CD86 molecules are surface glycoproteins and members of the immunoglobulin superfamily, which are expressed only on professional APCs (138-140). The blocking of this additional costimulatory signal leads to T cell anergy (141).

We reported that CD86 molecules play a prominent role in the antigen-specific induction of CD8 + CTLs when delivered as vaccine adjuvants (131). Coadministration of CD86 cDNA along with DNA-encoding HIV-1 antigens intramuscularly dramatically increased antigen-specific T cell responses without a significant change to the level of the humoral response. This enhancement of CTL response was both MHC class I restricted and CD8 + T cell dependent. Similar results have been obtained by other investigators who also found that CD86, not CD80, coexpression results in the enhancement of T cell-mediated immune responses (133,134).

Accordingly, we speculated that engineering of nonprofessional APCs such as muscle cells to express CD86 costimula-tory molecules could empower them to prime CTL precursors. However, the enhancement effect of CD86 codelivery could also have been mediated through the direct transfection of a small number of professional APCs residing within the muscle tissue. Subsequently, these cells could have greater expression of costimulatory molecules and could in theory become more potent. To investigate this issue, we constructed a set of bone marrow chimeric animals between normal mice and mice bearing a disrupted ^-microglobulin (p2m) gene (142). These bone marrow chimeras could respond and develop functional CTL responses following immunization with vaccinia virus. Next, we immunized chimeric animals with a DNA vaccine expressing HIV-1MN envelope protein (pCEnv) and plasmids encoding CD80 or CD86 genes (pCD80 or pCD86). Using this model, we observed that in vivo transfection of only pCEnv and pCD86 could engineer non-bone marrow-derived cells such as muscle cells to prime and expand CTLs. This study suggests that CD86 and not CD80 plays a central role in the generation of the antigen-specific CTL responses. These results indicate that the strategy of engineering muscle cells to be more efficient APCs could be an important tool for the optimization of antigen-specific T cell-mediated immune responses in a pursuit of more rationally designed vaccines and immune therapies through the control of MHC class I restriction. This method of engineering nonhematopoietic cells to be more efficient APCs could be especially important in cases where antigen alone fails to elicit a CTL response due to poor presentation by the host APCs.

In addition to the B7 family of costimulatory molecules, the immunology of CD40 and its ligand has also been implemented to enhance vaccine potency. CD40 functions by interacting with CD40 ligand (CD40L) expressed on activated CD4+ or CD8+ T cells. The attachment of CD40L onto APCs, with or without T helper cells, has been shown to ''condition'' the APCs for antigen-specific CTL activation (143-145). In addition, their ligation has shown to enhance the expression of B7 costimulatory molecules on APCs, including dendritic cells (146-148). Therefore, the engagement of CD40 with its ligand becomes advantageous for activation of CTLs during an immune priming response. Accordingly, coimmuni-zation of plasmids coding for p-galactosidase and CD40L has been reported to induce immune enhancement (149). Importantly, the addition induced enhancement of CTL responses without suppressing the development of antibody responses (150,151). Interestingly, coinjection of CD40L was revealed to be more effective than CD40 (152), indicating that expression of CD40L on muscle cells may also induce the ''licensing'' of APCs for the activation of CTLs. However, the possibility of direct transfection into antigen-specific infiltrating T cells cannot be eliminated, although T cells have not been yet established as an in vivo transfection target for DNA vaccines.

In light of these findings, we further investigated the strategy of engineering immune responses using additional costi-mulatory molecules (153). We coimmunized cDNA expression cassettes encoding intracellular adhesion molecule (ICAM)-1, lymphocyte function-associated antigen (LFA)-3, and vascular cell adhesion molecule (VCAM)-1, along with DNA immunogens, and analyzed the resulting antigen-specific immune responses. We observed that antigen-specific T cell responses can be enhanced by the coexpression of DNA immunogen and adhesion molecules ICAM-1 and LFA-3. Coexpression of ICAM-1 or LFA-3 molecules along with DNA immunogens resulted in a significant enhancement of Th cell proliferative responses. In addition, coimmunization with ICAM-1 (and more moderately with LFA-3) resulted in a dramatic enhancement of CD8-restricted CTL responses. Although VCAM-1 and ICAM-1 are similar in size, VCAM-1 coimmunization did not have any measurable effect on cellmediated responses. Rather, these results imply that ICAM-1 and LFA-3 provide direct T cell costimulation. These observations were further supported by the finding that coinjection with ICAM-1 dramatically enhanced the level of IFN-y and p-chemokines MIP-1a, MIP-1p, and RANTES produced by stimulated T cells. Through comparative studies, we observed that ICAM-1/LFA-1 T cell costimulatory pathways are independent of CD86/CD28 pathways, and they may synergisti-cally expand T cell responses in vivo. Furthermore, these studies indicate that CD8+ effector T cells at the site of inflammation can regulate the level of effector function through the expression of specific chemokines and adhesion molecules (Fig. 7) (130,153). Therefore, the end-stage effector T cells in the expansion phase of an antigen-specific immune response could direct their destiny through coordinated expression and release of these molecules.

D. Cell Death to Enhance Immunogenicity

In addition to powerful signaling, dendritic cells often function as scavenger cells by engulfing and processing apoptotic bodies. For instance, immature dendritic cells phagocytose apop-totic bodies by employing the receptors alphavbeta5 integrin and CD36 (154-156). Subsequent engulfment of the apoptotic body by both immature and mature dendritic cells induces viral and tumor immunogens to activate MHC class I restricted

Figure 7 Regulation of CD8 + T cell expansion by adhesion molecules and chemokines in the periphery. Specific adhesion molecules and chemokines provide modulatory signals to CD8 + T cells in effector stage. This network of cytokine, chemokine, costimulatory molecules and adhesion molecules represents a coordinated regulation and maintenance of effector T cells in the periphery.

Figure 7 Regulation of CD8 + T cell expansion by adhesion molecules and chemokines in the periphery. Specific adhesion molecules and chemokines provide modulatory signals to CD8 + T cells in effector stage. This network of cytokine, chemokine, costimulatory molecules and adhesion molecules represents a coordinated regulation and maintenance of effector T cells in the periphery.

CD8+ CTLs (17,156). Interestingly, in vivo depletion of CD11c + and CD8 + cells in mice fail to cross-prime antigens to prime naive T cells, specifying its essential role in inducing T cell activation (158). Both dendritic cells and macrophages have been shown to present apoptotic engulfed antigens, but the latter fails to activate naive T cells, which becomes a vital step in the activation of adaptive immunity (17,157). In addition, Rovere et al. and Ronchetti et al. demonstrated that there is a quantitative dependency on apoptotic bodies by dendritic cells in inducing the secretion of proinflammatory cyto-kines TNF-a and IL-1 p both in vitro and in vivo, respectively (156,159). Hence, an optimum strategy to develop potent vaccines would necessitate the activation of dendritic cells and the packaging of immunogens in these apoptotic bodies for uptake. Appropriately, we have employed a novel strategy whereby immunogen constructs were coimmunized with the death cell receptor Fas. In theory, this model induces the expression of both the immunogen and Fas within the same muscle cells and apoptosis would materialize through the interaction of Fas with its ligand, possibly via T cell assistance. When these 2 constructs were coimmunized, there was a significant augmentation of immune responses as measured by enhanced CTLs and Th1 cytokines, including IFN-y and IL-12 (160). In addition, implementing Fas as the apoptosis receptor may also provide a compounding effect on dendritic cell maturation, as Fas engagement possesses multiple functional roles. For instance, Fas not only contributes with the induction of cell death, but also stimulates the maturation of dendritic cells when engaged with its ligand (161). This is especially crucial, because direct in vivo transfection of dendritic cells has been proposed as a potential mechanism responsible for the induction of immune activation with respect to DNA vaccines (18-20). More recent work by Sasaki et al. implemented mutant caspases to decrease apoptotic efficiency to aliquot ample time for immunogen expression, while still delivering apoptosis-mediated antigens to dendritic cells (162). This raises an interesting question because these mutant caspases decreased apoptotic efficiency in vitro by nearly 10fold from the native caspase proteases (162). However, it is also currently understood that high quantities of apoptotic bodies are engulfed by scavenger macrophages and induce subsequent release of anti-inflammatory factors including IL-10, TGF-p, PGE2, and PAF (163,164). Accordingly, inflammation is observed when the clearance of apoptotic bodies becomes inefficient and results in their delayed removal from the surrounding environment (165). This results in the development of postapoptotic necrosis, which may function to deliver additional signals for proinflammatory developments and dendritic cell maturation (165,166). However, decrease in overall apoptotic quantity and potency with mutant caspases was able to provide enhanced immune response levels when compared with the more potent apoptotic signal (162). Therefore, it is also likely that delaying the expression of the apop-totic signal and/or working upstream of caspases may validate the requirement for the balance between immunogen expression vs. apoptotic stimuli (162). This reveals the necessity to delay the expression of apoptotic signals to maximize immunogen expression, while maintaining the potency of the death signal. It is also important to note that necrosis, not apoptosis, is traditionally the prime cell death mechanism by which inflammation and immune activation occurs (167). Interestingly, recent work suggests several models may be involved to induce immune activation through cell death. For instance, one model suggests that necrosis in conjunction with apoptosis delivers the maturation signal to dendritic cells (166,168). However, the role of postapoptotic necrosis as the stimulus is ruled out or insufficiently strong to deliver the maturation signal. Another model suggests that both primary necrotic and apoptotic cells are equivalent at inducing the maturation of dendritic cells and initiating immune activation (169). Therefore, either channel of cell death may be competent to stimulate the immune system for activation. Lastly, it is suggested that cell death or injury releases adjuvanting properties from the cytoplasm and is able to effectively stimulate immune activation (170). Therefore, components are actively secreted from dead cells that trigger the induction of immune responses. It is also evident that apoptosis and necrosis may function together to deliver immunogens to dendritic cells while inducing potent maturation signals (168). However, further studies are crucial to validate the precise roles that these cell death components may play in the generation of inflammatory responses with DNA immunizations.

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