Mamatha Seethammagari

An essential role for Akt1 in dendritic cell function and tumor immunotherapy

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, MF-ΔAkt, is sufficient for maturation and survival of murine bone marrow–derived DCs in vivo. MF-ΔAkt–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 MF-ΔhAkt 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.

Enhanced Activation of Human Dendritic Cells by Inducible CD40 and Toll-like Receptor-4 Ligation

Despite the potency of dendritic cells (DC) as antigen-presenting 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 tumor-associated antigens, including prostate-specific membrane antigen, and elimination of preestablished tumors, supporting this technology as a potent strategy for DC-based cancer immunotherapy.

Wnt and Notch Pathways Have Interrelated Opposing Roles on Prostate Progenitor Cell Proliferation and Differentiation

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. STEM CELLS 2011;29:678–688

FGFR1-WNT-TGF-β signaling in prostate cancer mouse models recapitulates human reactive stroma

The reactive stroma surrounding tumor lesions performs critical roles ranging from supporting tumor cell proliferation to inducing tumorigenesis and metastasis. Therefore, it is critical to understand the cellular components and signaling control mechanisms that underlie the etiology of reactive stroma. Previous studies have individually implicated fibroblast growth factor receptor 1 (FGFR1) and canonical WNT/β-catenin signaling in prostate cancer progression and the initiation and maintenance of a reactive stroma; however, both pathways are frequently found to be coactivated in cancer tissue. Using autochthonous transgenic mouse models for inducible FGFR1 (JOCK1) and prostate-specific and ubiquitously expressed inducible β-catenin (Pro-Cat and Ubi-Cat, respectively) and bigenic crosses between these lines (Pro-Cat × JOCK1 and Ubi-Cat × JOCK1), we describe WNT-induced synergistic acceleration of FGFR1-driven adenocarcinoma, associated with a pronounced fibroblastic reactive stroma activation surrounding prostatic intraepithelial neoplasia (mPIN) lesions found both in in situ and reconstitution assays. Both mouse and human reactive stroma exhibited increased transforming growth factor-β (TGF-β) signaling adjacent to pathologic lesions likely contributing to invasion. Furthermore, elevated stromal TGF-β signaling was associated with higher Gleason scores in archived human biopsies, mirroring murine patterns. Our findings establish the importance of the FGFR1-WNT-TGF-β signaling axes as driving forces behind reactive stroma in aggressive prostate adenocarcinomas, deepening their relevance as therapeutic targets.

EZC-Prostate Models Offer High Sensitivity and Specificity for Noninvasive Imaging of Prostate Cancer Progression and Androgen Receptor Action

In vivo imaging advances have greatly expanded the use of animal cancer models. Herein, we describe two new models that permit prostate imaging ex vivo, in vivo, and in utero. Further, we show the use of these models for detecting small metastasis and testing reagents that modulate the androgen receptor (AR) axis. A luciferase reporter gene was directed to the prostate epithelium using three composite promoters called human kallikrein 2 (hK2)-E3/P, PSA-E2/P, and ARR2PB, derived from hK2, PSA, and rat probasin regulatory elements, to generate the EZC1, EZC2, and EZC3-prostate mice, respectively. EZC2 and EZC3-prostate display robust expression in the prostate with only minimal detectable expression in other organs, including testes and epididymis. Luciferase expression was detected as early as embryonic day 13 (E13) in the urogenital track. To image prostate cancer progression, lines of EZC mice were bred with prostate cancer models TRAMP and JOCK1, and imaged longitudinally. When crossed with prostate cancer models, EZC3 facilitated detection of metastatic lesions although total prostate luciferase expression was static or reduced due to weakening of AR-regulated promoters. Castration reduced luciferase expression by 90% and 97% in EZC2 and EZC3 mice, respectively, and use of GnRH antagonist also led to extensive inhibition of reporter activity. The EZC-prostate model permits prostate imaging in vivo and should be useful for imaging prostate development, growth, metastasis, and response to treatment noninvasively and longitudinally. These models also provide powerful new reagents for developing improved drugs that inhibit the AR axis.

The Phosphatase Src Homology Region 2 Domain-Containing Phosphatase-1 Is an Intrinsic Central Regulator of Dendritic Cell Function

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.

Activation of Wnt Signaling by Chemically Induced Dimerization of LRP5 Disrupts Cellular Homeostasis

Wnt signaling is crucial for a variety of biological processes, including body axis formation, planar polarity, stem cell maintenance and cellular differentiation. Therefore, targeted manipulation of Wnt signaling in vivo would be extremely useful. By applying chemical inducer of dimerization (CID) technology, we were able to modify the Wnt co-receptor, low-density lipoprotein (LDL)-receptor-related protein 5 (LRP5), to generate the synthetic ligand inducible Wnt switch, iLRP5. We show that iLRP5 oligomerization results in its localization to disheveled-containing punctate structures and sequestration of scaffold protein Axin, leading to robust β-catenin-mediated signaling. Moreover, we identify a novel LRP5 cytoplasmic domain critical for its intracellular localization and casein kinase 1-dependent β-catenin signaling. Finally, by utilizing iLRP5 as a Wnt signaling switch, we generated the Ubiquitous Activator of β-catenin (Ubi-Cat) transgenic mouse line. The Ubi-Cat line allows for nearly ubiquitous expression of iLRP5 under control of the H-2Kb promoter. Activation of iLRP5 in isolated prostate basal epithelial stem cells resulted in expansion of p63+ cells and development of hyperplasia in reconstituted murine prostate grafts. Independently, iLRP5 induction in adult prostate stroma enhanced prostate tissue regeneration. Moreover, induction of iLRP5 in male Ubi-Cat mice resulted in prostate tumor progression over several months from prostate hyperplasia to adenocarcinoma. We also investigated iLRP5 activation in Ubi-Cat-derived mammary cells, observing that prolonged activation results in mammary tumor formation. Thus, in two distinct experimental mouse models, activation of iLRP5 results in disruption of tissue homeostasis, demonstrating the utility of iLRP5 as a novel research tool for determining the outcome of Wnt activation in a precise spatially and temporally determined fashion.

Tobacco mosaic virus efficiently targets DC uptake, activation and antigen-specific T cell responses in vivo.

Over the past 20 years, dendritic cells (DCs) have been utilized to activate immune responses capable of eliminating cancer cells. Currently, ex vivo DC priming has been the mainstay of DC cancer immunotherapies. However, cell-based treatment modalities are inherently flawed due to a lack of standardization, specialized facilities and personnel, and cost. Therefore, direct modes of DC manipulation, circumventing the need for ex vivo culture, must be investigated. To facilitate the development of next-generation, in vivo targeted DC vaccines, we characterized the DC interaction and activation potential of the Tobacco Mosaic virus (TMV), a plant virus that enjoys a relative ease of production and the ability to deliver protein payloads via surface conjugation. In this study we show that TMV is readily taken up by mouse bone marrow-derived DCs, in vitro. Footpad injection of fluorophore-labeled TMV reveals preferential uptake by draining lymph node resident DCs in vivo. Uptake leads to activation, as measured by the upregulation of key DC surface markers. When peptide antigen-conjugated TMV is injected into the footpad of mice, DC-mediated uptake and activation leads to robust antigen-specific CD8(+) T cell responses, as measured by antigen-specific tetramer analysis. Remarkably, TMV priming induced a greater magnitude T cell response than Adenovirus (Ad) priming. Finally, TMV is capable of boosting either Ad-induced or TMV-induced antigen-specific T cell responses, demonstrating that TMV, uniquely, does not induce neutralizing self-immunity. Overall, this study elucidates the in vivo DC delivery and activation properties of TMV and indicates its potential as a vaccine vector in stand alone or prime-boost strategies.

Off-the-shelf Adenoviral-mediated Immunotherapy via Bicistronic Expression of Tumor Antigen and iMyD88/CD40 Adjuvant

Recent modest successes in ex vivo dendritic cell (DC) immunotherapy have motivated continued innovation in the area of DC manipulation and activation. Although ex vivo vaccine approaches continue to be proving grounds for new DC manipulation techniques, the intrinsic limits of ex vivo therapy, including high cost, minimal standardization, cumbersome delivery, and poor accessibility, incentivizes the development of vaccines compatible with in vivo DC targeting. We describe here a method to co-deliver both tumor-specific antigen (TSA) and an iMyD88/CD40 adjuvant (iMC), to DCs that combines toll-like receptor (TLR) and CD40 signaling. In this study, we demonstrate that simple TSA delivery via adenoviral vectors results in strong antitumor immunity. Addition of iMC delivered in a separate vector is insufficient to enhance this effect. However, when delivered simultaneously with TSA in a single bicistronic vector (BV), iMC is able to significantly enhance antigen-specific cytotoxic T-cell (CTL) responses and inhibit established tumor growth. This study demonstrates the spatial-temporal importance of concurrent DC activation and TSA presentation. Further, it demonstrates the feasibility of in vivo molecular enhancement of DCs necessary for effective antitumor immune responses.

The E3 ligase c‐Cbl regulates dendritic cell activation

The activation of innate and adaptive immunity is always balanced by inhibitory signalling mechanisms to maintain tissue integrity. We have identified the E3 ligase c‐Cbl––known for its roles in regulating lymphocyte signalling––as a modulator of dendritic cell activation. In c‐Cbl‐deficient dendritic cells, Toll‐like receptor‐induced expression of proinflammatory factors, such as interleukin‐12, is increased, correlating with a greater potency of dendritic‐cell‐based vaccines against established tumours. This proinflammatory phenotype is accompanied by an increase in nuclear factor (NF)‐κB activity. In addition, c‐Cbl deficiency reduces both p50 and p105 levels, which have been shown to modulate the stimulatory function of NF‐κB. Our data indicate that c‐Cbl has a crucial, RING‐domain‐dependent role in regulating dendritic cell maturation, probably by facilitating the regulatory function of p105 and/or p50.