Current dendritic cell (DC) vaccine preparations involving ex vivo differentiation and maturation produce short-lived, transiently active DCs that may curtail T-cell responses in vivo. We demonstrate that Akt1, downregulation of which decreases DC lifespan, is critical for proinflammatory signal-mediated DC survival and maturation. Lipopolysaccharide or CD40 signaling stabilizes Akt1, promoting both activation and Bcl-2-dependent survival of DCs. Expression of a potent allele encoding a lipid raft-targeted Akt1, M(F)-DeltaAkt, is sufficient for maturation and survival of murine bone marrow-derived DCs in vivo. M(F)-DeltaAkt-transduced DCs enhanced T-cell proliferation, activation and long-term memory responses, enabling eradication of large pre-established lymphomas and aggressive B16 melanomas. Human myeloid DCs expressing constitutively active M(F)-DeltahAkt also survived significantly longer and promoted antigen-specific T-cell responses. Thus, Akt1 is a critical regulator of DC lifespan, and its manipulation in DCs can improve the clinical efficacy of DC-based tumor vaccines.

The in vivo therapeutic efficacy of DC-based cancer vaccines is limited by suboptimal DC maturation protocols. Although delivery of TLR adjuvants systemically boosts DC-based cancer vaccine efficacy, it could also increase toxicity. Here, we have engineered a drug-inducible, composite activation receptor for DCs (referred to herein as DC-CAR) comprising the TLR adaptor MyD88, the CD40 cytoplasmic region, and 2 ligand-binding FKBP12 domains. Administration of a lipid-permeant dimerizing ligand (AP1903) induced oligomerization and activation of this fusion protein, which we termed iMyD88/CD40. AP1903 administration to vaccinated mice enabled prolonged and targeted activation of iMyD88/CD40-modified DCs. Compared with conventionally matured DCs, AP1903-activated iMyD88/CD40-DCs had increased activation of proinflammatory MAPKs. AP1903-activated iMyD88/CD40-transduced human or mouse DCs also produced higher levels of Th1 cytokines, showed improved migration in vivo, and enhanced both antigen-specific CD8 + T cell responses and innate NK cell responses. Furthermore, treatment with AP1903 in vaccinated mice led to robust antitumor immunity against preestablished E.G7-OVA lymphomas and aggressive B16.F10 tumors. Thus, the iMyD88/ CD40 unified "switch" effectively and safely replaced exogenous adjuvant cocktails, allowing remote and sustained DC activation in vivo. DC "licensing" through iMyD88/CD40 may represent a mechanism by which to exploit the natural synergy between the TLR and CD40 signaling pathways in DCs using a single small molecule drug and could augment the efficacy of antitumor DC-based vaccines.

Despite the potency of dendritic cells (DC) as antigenpresenting cells for priming adaptive immunity, DC-based cancer vaccines have been largely insufficient to effectively reduce tumor burden or prevent tumor progression in most patients. To enhance DC-based vaccines, we used the combination of a synthetic ligand-inducible CD40 receptor (iCD40) along with Toll-like receptor-4 (TLR-4) ligation in human monocyte-derived DCs. The iCD40 receptor permits targeted, reversible activation of CD40 in vivo, potentially bypassing the essential role of CD4 + T cells for activation of DCs. As a rigorous preclinical study of this approach, we evaluated key parameters of DC activation and function. Whereas neither iCD40 nor TLR-4 signaling alone led to high levels of interleukin (IL)-12p70 and IL-6, using iCD40 in combination with lipopolysaccharide (LPS) or monophosphoryl lipid A led to strongly synergistic production of both. Furthermore, this approach led to high expression of DC maturation markers, epitope-specific CTL and T helper 1 responses, as well as DC migration in vitro and in vivo. Moreover, use of iCD40-modified and LPS-stimulated DCs led to targeted expansion of autologous T cells against tumorassociated antigens, including prostate-specific membrane antigen, and elimination of preestablished tumors, supporting this technology as a potent strategy for DC-based cancer immunotherapy.

Dendritic cells (DCs) initiate proinflammatory or regulatory T cell responses, depending on their activation state. Despite extensive knowledge of DC-activating signals, the understanding of DC inhibitory signals is relatively limited. We show that Src homology region 2 domain-containing phosphatase-1 (SHP-1) is an important inhibitor of DC signaling, targeting multiple activation pathways. Downstream of TLR4, SHP-1 showed increased interaction with several proteins including IL-1R–associated kinase-4, and modulated LPS signaling by inhibiting NF-κB, AP-1, ERK, and JNK activity, while enhancing p38 activity. In addition, SHP-1 inhibited prosurvival signaling through AKT activation. Furthermore, SHP-1 inhibited CCR7 protein expression. Inhibiting SHP-1 in DCs enhanced proinflammatory cytokines, IL-6, IL-12, and IL-1β production, promoted survival, and increased DC migration to draining lymph nodes. Administration of SHP-1–inhibited DCs in vivo induced expansion of Ag-specific cytotoxic T cells and inhibited Foxp3+ regulatory T cell induction, resulting in an enhanced immune response against pre-established mouse melanoma and prostate tumors. Taken together, these data demonstrate that SHP-1 is an intrinsic global regulator of DC function, controlling many facets of T cell-mediated immune responses.

Tissue stem cells are capable of both self-renewal and differentiation to maintain a constant stem cell population and give rise to the plurality of cells within a tissue. Wnt signaling has been previously identified as a key mediator for the maintenance of tissue stem cells; however, possible cross-regulation with other developmentally critical signaling pathways involved in adult tissue homeostasis, such as Notch, is not well understood. By using an in vitro prostate stem cell colony (“prostasphere”) formation assay and in vivo prostate reconstitution experiments, we demonstrate that Wnt pathway induction on Sca-1+ CD49f+ basal/stem cells (B/SCs) promotes expansion of the basal epithelial compartment with noticeable increases in “triple positive” (cytokeratin [CK] 5+, CK8+, p63+) prostate progenitor cells, concomitant with upregulation of known Wnt target genes involved in cell-cycle induction. Moreover, Wnt induction affects expression of epithelial-to-mesenchymal transition signature genes, suggesting a possible mechanism for priming B/SC to act as potential tumor-initiating cells. Interestingly, induction of Wnt signaling in B/SCs results in downregulation of Notch1 transcripts, consistent with its postulated antiproliferative role in prostate cells. In contrast, induction of Notch signaling in prostate progenitors inhibits their proliferation and disrupts prostasphere formation. In vivo prostate reconstitution assays further demonstrate that induction of Notch in B/SCs disrupts proper acini formation in cells expressing the activated Notch1 allele, Notch-1 intracellular domain. These data emphasize the importance of Wnt/Notch cross-regulation in adult stem cell biology and suggest that Wnt signaling controls the proliferation and/or maintenance of epithelial progenitors via modulation of Notch signaling.