Efficient dendritic cell priming of T lymphocytes depends on the extracellular matrix protein mindin

Rho guanosine triphosphatases (GTPases) regulate multiple aspects of dendritic cell (DC) function, but what regulates the expression of Rho GTPases in DCs is unknown. Here, we show that the extracellular matrix protein mindin regulates the expression of Rho GTPases in DCs. Mindin(-/-) mice displayed defective CD4+ T-cell priming and impaired humoral immune responses to T-dependent antigens. Mindin(-/-) DCs had reduced expression of Rac1/2 and impaired priming capacity owing to inefficient engagement with T lymphocytes. Ectopic Rac1 expression restored the priming capability of Mindin(-/-) DCs. Furthermore, we show that DC adhesion to mindin matrix was blocked by antibodies to alpha4, alpha5 and beta1 integrins. DCs lacking beta1 integrin had reduced adhesion to mindin matrix, decreased expression of Rac1/2 and impaired priming capacity. These results suggest that mindin-integrin interactions play a key role in regulating Rho GTPase expression in DCs and DC priming of T lymphocytes.     

Acetylcorynoline Impairs the Maturation of Mouse Bone Marrow-Derived Dendritic Cells via Suppression of IκB Kinase and Mitogen-Activated Protein Kinase Activities

Background

Dendritic cells (DCs) are major modulators in the immune system. One active field of research is the manipulation of DCs as pharmacological targets to screen novel biological modifiers for the treatment of inflammatory and autoimmune disorders. Acetylcorynoline is the major alkaloid component derived from Corydalis bungeana herbs. We assessed the capability of acetylcorynoline to regulate lipopolysaccharide (LPS)-stimulated activation of mouse bone marrow-derived DCs.

Methodology/Principal Findings

Our experimental data showed that treatment with up to 20 µM acetylcorynoline does not cause cytotoxicity in cells. Acetylcorynoline significantly inhibited the secretion of tumor necrosis factor-α, interleukin-6, and interleukin-12p70 by LPS-stimulated DCs. The expression of LPS-induced major histocompatibility complex class II, CD40, and CD86 on DCs was also decreased by acetylcorynoline, and the endocytic capacity of LPS-stimulated DCs was restored by acetylcorynoline. In addition, LPS-stimulated DC-elicited allogeneic T-cell proliferation was blocked by acetylcorynoline, and the migratory ability of LPS-stimulated DCs was reduced by acetylcorynoline. Moreover, acetylcorynoline significantly inhibits LPS-induced activation of IκB kinase and mitogen-activated protein kinase. Importantly, administration of acetylcorynoline significantly attenuates 2,4-dinitro-1-fluorobenzene-induced delayed-type hypersensitivity.

Conclusions/Significance

Acetylcorynoline may be one of the potent immunosuppressive agents through the blockage of DC maturation and function.     

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, 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.     

Effect of oxygen levels on the physiology of dendritic cells: implications for adoptive cell therapy

Dendritic cell (DC)-based adoptive tumor immunotherapy approaches have shown promising results, but the incidence of tumor regression is low and there is an evident call for identifying culture conditions that produce DCs with a more potent Th1 potential. Routinely, DCs are differentiated in CO(2) incubators under atmospheric oxygen conditions (21% O(2)), which differ from physiological oxygen levels of only 3-5% in tissue, where most DCs reside. We investigated whether differentiation and maturation of DCs under physiological oxygen levels could produce more potent T-cell stimulatory DCs for use in adoptive immunotherapy. We found that immature DCs differentiated under physiological oxygen levels showed a small but significant reduction in their endocytic capacity. The different oxygen levels did not influence their stimuli-induced upregulation of cluster of differentiation 54 (CD54), CD40, CD83, CD86, C-C chemokine receptor type 7 (CCR7), C-X-C chemokine receptor type 4 (CXCR4) and human leukocyte antigen (HLA)-DR or the secretion of interleukin (IL)-6, tumor necrosis factor (TNF)-α and IL-10 in response to lipopolysaccharide (LPS) or a cytokine cocktail. However, DCs differentiated under physiological oxygen level secreted higher levels of IL-12(p70) after exposure to LPS or CD40 ligand. Immature DCs differentiated at physiological oxygen levels caused increased T-cell proliferation, but no differences were observed for mature DCs with regard to T-cell activation. In conclusion, we show that although DCs generated under atmospheric or physiological oxygen conditions are mostly similar in function and phenotype, DCs differentiated under physiological oxygen secrete larger amounts of IL-12(p70). This result could have implications for the use of ex vivo-generated DCs for clinical studies, since DCs differentiated at physiological oxygen could induce increased Th1 responses in vivo.     

Influenza virus evades innate and adaptive immunity via the NS1 protein

Both antibodies and T cells contribute to immunity against influenza virus infection. However, the generation of strong Th1 immunity is crucial for viral clearance. Interestingly, we found that human dendritic cells (DCs) infected with influenza A virus have lower allospecific Th1-cell stimulatory abilities than DCs activated by other stimuli, such as lipopolysaccharide and Newcastle disease virus infection. This weak stimulatory activity correlates with a suboptimal maturation of the DCs following infection with influenza A virus. We next investigated whether the influenza A virus NS1 protein could be responsible for the low levels of DC maturation after influenza virus infection. The NS1 protein is an important virulence factor associated with the suppression of innate immunity via the inhibition of type I interferon (IFN) production in infected cells. Using recombinant influenza and Newcastle disease viruses, with or without the NS1 gene from influenza virus, we found that the induction of a genetic program underlying DC maturation, migration, and T-cell stimulatory activity is specifically suppressed by the expression of the NS1 protein. Among the genes affected by NS1 are those coding for macrophage inflammatory protein 1beta, interleukin-12 p35 (IL-12 p35), IL-23 p19, RANTES, IL-8, IFN-alpha/beta, and CCR7. These results indicate that the influenza A virus NS1 protein is a bifunctional viral immunosuppressor which inhibits innate immunity by preventing type I IFN release and inhibits adaptive immunity by attenuating human DC maturation and the capacity of DCs to induce T-cell responses. Our observations also support the potential use of NS1 mutant influenza viruses as live attenuated influenza virus vaccines.     

Dendritic cell transmigration through brain microvessel endothelium is regulated by MIP-1alpha chemokine and matrix metalloproteinases

Dendritic cells (DCs) accumulate in the CNS during inflammatory diseases, but the exact mechanism regulating their traffic into the CNS remains to be defined. We now report that MIP-1alpha increases the transmigration of bone marrow-derived, GFP-labeled DCs across brain microvessel endothelial cell monolayers. Furthermore, occludin, an important element of endothelial tight junctions, is reorganized when DCs migrate across brain capillary endothelial cell monolayers without causing significant changes in the barrier integrity as measured by transendothelial electrical resistance. We show that DCs produce matrix metalloproteinases (MMP) -2 and -9 and GM6001, an MMP inhibitor, decreases both baseline and MIP-1alpha-induced DC transmigration. These observations suggest that DC transmigration across brain endothelial cell monolayers is partly MMP dependent. The migrated DCs express higher levels of CD40, CD80, and CD86 costimulatory molecules and induce T cell proliferation, indicating that the transmigration of DCs across brain endothelial cell monolayers contributes to the maintenance of DC Ag-presenting function. The MMP dependence of DC migration across brain endothelial cell monolayers raises the possibility that MMP blockers may decrease the initiation of T cell recruitment and neuroinflammation in the CNS.     

Toll-Like Receptor 4 Engagement Drives Differentiation of Human and Murine Dendritic Cells from a Pro- into an Anti-Inflammatory Mode

The dendritic cell (DC) coordinates innate and adaptive immunity to fight infections and cancer. Our observations reveal that DCs exposed to the microbial danger signal lipopolysaccharide (LPS) in the presence of interferon-γ (IFN-γ) acquire a continuously changing activation/maturation phenotype. The DCs’ initial mode of action is pro-inflammatory via up-regulation among others of the signaling molecule interleukin (IL) 12, which polarizes IFN-γ secreting type 1 helper T-cells (Th1). Within 24 hours the same DC switches from the pro- into an anti-inflammatory phenotype. This is mediated by autocrine IL-10 release and secretion of soluble IL-2 receptor alpha (sIL-2RA) molecules. T-cells, when contacted with DCs during their anti-inflammatory phase loose their proliferative capacity and develop regulatory T-cell (Treg) -like anti-inflammatory functions indicated by IL-10 secretion and elevated FoxP3 levels. Studying the kinetics of IL-12 and IL-10 expression from LPS/IFN-γ activated myeloid DCs on a single cell level confirmed these observations. When T-cells are separated from DCs within 24 hours, they are spared from the anti-inflammatory DC activity. We conclude that, in addition to differentiation of DCs into distinct subsets, the observed sequential functional phases of DC differentiation permit the fine-tuning of an immune response. A better understanding of time-kinetic DC features is required for optimally exploiting the therapeutic capacity of DCs in cancer immune therapy.     

A critical role for prostaglandin E2 in podosome dissolution and induction of high-speed migration during dendritic cell maturation

Dendritic cells (DCs) are professional APCs of the immune system that play a key role in regulating T cell-based immunity. The capacity of DCs to activate T cells depends on their maturation state as well as their ability to migrate to the T cell areas of draining lymph nodes. In this study, we investigated the effects of DC maturation stimuli on the actin cytoskeleton and beta(1) integrin-dependent adhesion and migration. Podosomes, specialized adhesion structures found in immature monocyte-derived DCs as well as myeloid DCs, rapidly dissolve in response to maturation stimuli such as TNF-alpha and PGE(2), whereas the TLR agonist LPS induces podosome dissolution only after a long lag time. We demonstrate that LPS-mediated podosome disassembly as well as the onset of high-speed DC migration are dependent on the production of PGs by the DCs. Moreover, both of these processes are inhibited by Ab-induced activation of beta(1) integrins. Together, these results show that maturation-induced podosome dissolution and loss of alpha(5)beta(1) integrin activity allow human DCs to undergo the transition from an adhesive to a highly migratory phenotype.     

Carbomer-based adjuvant elicits CD8 T-cell immunity by inducing a distinct metabolic state in cross-presenting dendritic cells

There is a critical need for adjuvants that can safely elicit potent and durable T cell-based immunity to intracellular pathogens. Here, we report that parenteral vaccination with a carbomer-based adjuvant, Adjuplex (ADJ), stimulated robust CD8 T-cell responses to subunit antigens and afforded effective immunity against respiratory challenge with a virus and a systemic intracellular bacterial infection. Studies to understand the metabolic and molecular basis for ADJ’s effect on antigen cross-presentation by dendritic cells (DCs) revealed several unique and distinctive mechanisms. ADJ-stimulated DCs produced IL-1β and IL-18, suggestive of inflammasome activation, but in vivo activation of CD8 T cells was unaffected in caspase 1-deficient mice. Cross-presentation induced by TLR agonists requires a critical switch to anabolic metabolism, but ADJ enhanced cross presentation without this metabolic switch in DCs. Instead, ADJ induced in DCs, an unique metabolic state, typified by dampened oxidative phosphorylation and basal levels of glycolysis. In the absence of increased glycolytic flux, ADJ modulated multiple steps in the cytosolic pathway of cross-presentation by enabling accumulation of degraded antigen, reducing endosomal acidity and promoting antigen localization to early endosomes. Further, by increasing ROS production and lipid peroxidation, ADJ promoted antigen escape from endosomes to the cytosol for degradation by proteasomes into peptides for MHC I loading by TAP-dependent pathways. Furthermore, we found that induction of lipid bodies (LBs) and alterations in LB composition mediated by ADJ were also critical for DC cross-presentation. Collectively, our model challenges the prevailing metabolic paradigm by suggesting that DCs can perform effective DC cross-presentation, independent of glycolysis to induce robust T cell-dependent protective immunity to intracellular pathogens. These findings have strong implications in the rational development of safe and effective immune adjuvants to potentiate robust T-cell based immunity.     

Recombinant tumor-associated MUC1 glycoprotein impairs the differentiation and function of dendritic cells

Tumors exploit several strategies to evade immune recognition, including the production of a large number of immunosuppressive factors, which leads to reduced numbers and impaired functions of dendritic cells (DCs) in the vicinity of tumors. We have investigated whether a mucin released by tumor cells could be involved in causing these immunomodulating effects on DCs. We used a recombinant purified form of the MUC1 glycoprotein, an epithelial associated mucin that is overexpressed, aberrantly glycosylated, and shed during cancer transformation. The O-glycosylation profile of the recombinant MUC1 glycoprotein (ST-MUC1) resembled that expressed by epithelial tumors in vivo, consisting of large numbers of sialylated core 1 (sialyl-T, ST) oligosaccharides. When cultured in the presence of ST-MUC1, human monocyte-derived DCs displayed a modified phenotype with decreased expression of costimulatory molecules (CD86, CD40), Ag-presenting molecules (DR and CD1d), and differentiation markers (CD83). In contrast, markers associated with an immature phenotype, CD1a and CD206 (mannose receptor), were increased. This effect was already evident at day 4 of DC culture and was dose dependent. The modified phenotype of DCs corresponded to an altered balance in IL-12/IL-10 cytokine production, with DC expressing an IL-10(high)IL-12(low) phenotype after exposure to ST-MUC1. These DCs were defective in their ability to induce immune responses in both allogeneic and autologous settings, as detected in proliferation and ELISPOT assays. The altered DC differentiation and Ag presentation function induced by the soluble sialylated tumor-associated mucin may represent a mechanism by which epithelial tumors can escape immunosurveillance.     

Selective serotonin reuptake inhibitors attenuate the antigen presentation from dendritic cells to effector T lymphocytes

Fluoxetine, one of the selective serotonin reuptake inhibitors (SSRIs), has been found to possess immune modulation effects, in addition to its antidepressant effects. However, it remains unclear whether SSRIs can suppress the antigen-presenting function of dendritic cells (DCs). Therefore, Fluoxetine was applied to a co-culture of Aggregatibacter actinomycetemcomitans (Aa)-reactive T cells (×Aa-T) isolated from Aa-immunized mice and DCs. This resulted in the suppressed proliferation of ×Aa-T stimulated with Aa-antigen presentation by DCs. Specifically, Fluoxetine increased the extracellular 5-hydroxytryptamine (5-HT) in the ×Aa-T/DC co-culture, whereas exogenously applied 5-HT promoted T-cell proliferation in the ×Aa-T/DC co-culture, indicating that Fluoxetine-mediated suppression of ×Aa-T/DC responses cannot be attributed to extracellular 5-HT. Instead, Fluoxetine remarkably suppressed the expression of costimulatory molecule ICOS-L on DCs. Fluoxetine also promoted a greater proportion of CD86(Low) immature DCs than CD86(High) mature DCs, while maintaining the expression levels of CD80, MHC-class-II and PD-L1. These results suggested that Fluoxetine suppressed the ability of DCs to present bacterial antigens to T cells, and the resulting T-cell proliferation, in a SERT/5-HT-independent manner and that diminished expression of ICOS-L on DCs and increase of CD86(Low) immature DCs caused by Fluoxetine might be partially associated with Fluoxetine-mediated suppression of DC/T-cell responses.     

Recognition of enteroinvasive Escherichia coli and Shigella flexneri by dendritic cells: distinct dendritic cell activation states

The innate and adaptive immune responses of dendritic cells (DCs) to enteroinvasive Escherichia coli (EIEC) infection were compared with DC responses to Shigella flexneri infection. EIEC triggered DCs to produce interleukin (IL)-10, IL-12 and tumour necrosis factor (TNF)-α, whereas S. flexneri induced only the production of TNF-α. Unlike S. flexneri, EIEC strongly increased the expression of toll like receptor (TLR)-4 and TLR-5 in DCs and diminished the expression of co-stimulatory molecules that may cooperate to inhibit CD4(+) T-lymphocyte proliferation. The inflammation elicited by EIEC seems to be related to innate immunity both because of the aforementioned results and because only EIEC were able to stimulate DC transmigration across polarised Caco-2 cell monolayers, a mechanism likely to be associated with the secretion of CC chemokine ligands (CCL)20 and TNF-α. Understanding intestinal DC biology is critical to unravelling the infection strategies of EIEC and may aid in the design of treatments for infectious diseases.     

Specific microtubule-depolymerizing agents augment efficacy of dendritic cell-based cancer vaccines

Background

Damage-associated molecular patterns (DAMPs) are associated with immunogenic cell death and have the ability to enhance maturation and antigen presentation of dendritic cells (DCs). Specific microtubule-depolymerizing agents (MDAs) such as colchicine have been shown to confer anti-cancer activity and also trigger activation of DCs.

Methods

In this study, we evaluated the ability of three MDAs (colchicine and two 2-phenyl-4-quinolone analogues) to induce immunogenic cell death in test tumor cells, activate DCs, and augment T-cell proliferation activity. These MDAs were further evaluated for use as an adjuvant in a tumor cell lysate-pulsed DC vaccine.

Results

The three test phytochemicals considerably increased the expression of DAMPs including HSP70, HSP90 and HMGB1, but had no effect on expression of calreticulin (CRT). DC vaccines pulsed with MDA-treated tumor cell lysates had a significant effect on tumor growth, showed cytotoxic T-lymphocyte activity against tumors, and increased the survival rate of test mice. In vivo antibody depletion experiments suggested that CD8⁺ and NK cells, but not CD4⁺ cells, were the main effector cells responsible for the observed anti-tumor activity. In addition, culture of DCs with GM-CSF and IL-4 during the pulsing and stimulation period significantly increased the production of IL-12 and decreased production of IL-10. MDAs also induced phenotypic maturation of DCs and augmented CD4⁺ and CD8⁺ T-cell proliferation when co-cultured with DCs.

Conclusions

Specific MDAs including the clinical drug, colchicine, can induce immunogenic cell death in tumor cells, and DCs pulsed with MDA-treated tumor cell lysates (TCLs) can generate potent anti-tumor immunity in mice. This approach may warrant future clinical evaluation as a cancer vaccine.     

The ischemia-responsive protein 94 (Irp94) activates dendritic cells through NK cell receptor protein-2/NK group 2 member D (NKR-P2/NKG2D) leading to their maturation

Tumor recognition and killing, the uptake of released immunogenic substrate, and the generation of immunity are crucial aspects of dendritic cell (DC)-mediated antitumor immune response. In the context of direct tumoricidal activity, we have recently shown NK cell receptor protein-2 (NKR-P2)/NK group 2 member D (NKG2D) as a potent activation receptor on rat DCs. The activation of DCs with agonistic anti-NKR-P2 mAb, the binding of soluble NKR-P2 to the AK-5 tumor, and DC maturation with fixed AK-5 cells led us to identify a putative NKR-P2 ligand on the AK-5 cell surface. In this study we have shown that the AK-5 tumor-derived ischemia-responsive protein-94 (Irp94, a 110 kDa Hsp family member) acts as a functional ligand for NKR-P2 on DCs and enhances Irp94-NKR-P2 interaction-dependent tumor cell apoptosis via NO. Surface expression of Irp94 was also found on tumors of diverse origin in addition to AK-5. Furthermore, the Th1-polarizing cytokine IL-12, produced from Irp94-ligated BMDCs, augments NK cell cytotoxicity. Irp94-NKR-P2 interaction drives the maturation of BMDCs by up-regulating MHC class II, CD86, and CD1a and also induces autologous T cell proliferation, which displays a crucial state of DCs for adaptive antitumor immune response. These functional properties of Irp94 reside in the COOH terminus subdomain but not in the NH2 terminus ATPase domain of Irp94. We also show the involvement of PI3K, ERK, protein kinase C, phosphatases, and NF-kappaB translocation as downstream mediators of DCs activation upon NKR-P2 ligation with Irp94. Our studies demonstrate for the first time a novel role of a 110-kDa heat shock protein (Irp94) as a ligand for NKR-P2 on DCs, which in turn executes both innate and adaptive immunity.     

Whole recombinant yeast vaccine activates dendritic cells and elicits protective cell-mediated immunity

There is currently a need for vaccines that stimulate cell-mediated immunity-particularly that mediated by CD8+ cytotoxic T lymphocytes (CTLs)-against viral and tumor antigens. The optimal induction of cell-mediated immunity requires the presentation of antigens by specialized cells of the immune system called dendritic cells (DCs). DCs are unique in their ability to process exogenous antigens via the major histocompatibility complex (MHC) class I pathway as well as in their ability to activate naive, antigen-specific CD8+ and CD4+ T cells. Vaccine strategies that target or activate DCs in order to elicit potent CTL-mediated immunity are the subject of intense research. We report here that whole recombinant Saccharomyces cerevisiae yeast expressing tumor or HIV-1 antigens potently induced antigen-specific, CTL responses, including those mediating tumor protection, in vaccinated animals. Interactions between yeast and DCs led to DC maturation, IL-12 production and the efficient priming of MHC class I- and class II-restricted, antigen-specific T-cell responses. Yeast exerted a strong adjuvant effect, augmenting DC presentation of exogenous whole-protein antigen to MHC class I- and class II-restricted T cells. Recombinant yeast represent a novel vaccine strategy for the induction of broad-based cellular immune responses.     

Day-4 myeloid dendritic cells pulsed with whole tumor lysate are highly immunogenic and elicit potent anti-tumor responses

"Day-7" myeloid DCs are commonly used in the clinic. However, there is a strong need to develop DCs faster that have the same potent immunostimulatory capacity as "Day-7" myeloid DCs and at the same time minimizing time, labor and cost of DC preparations. Although "2 days" DCs can elicit peptide-specific responses, they have not been demonstrated to engulf, process and present complex whole tumor lysates, which could be more convenient and personalized source of tumor antigens than defined peptides. In this preclinical study, we evaluated the T-cell stimulatory capacity of Day-2, Day-4, and Day-7 cultured monocyte-derived DCs loaded with SKOV3 cell whole lysate prepared by freeze-thaw or by UVB-irradiation followed by freeze-thaw, and matured with lipopolysaccharide (LPS) and interferon (IFN)-gamma. DCs were evaluated for antigen uptake, and following maturation with LPS and IFN-gamma, DCs were assessed for expression of CD80, CD40, CD86, ICAM-1 and CCR7, production of IL-12p70 and IP-10, and induction of tumor-specific T-cell responses. Day-4 and Day-7 DCs exhibited similar phagocytic abilities, which were superior to Day-2 DCs. Mature Day-7 DCs expressed the highest CD40 and ICAM-1, but mature Day-4 DCs produced the most IL-12p70 and IP-10. Importantly, Day-4 and Day-7 DCs derived from ovarian cancer patients stimulated equally strongly tumor-specific T-cell responses. This is the first study demonstrating the highly immunogenic and strong T-cell stimulatory properties of Day-4 myeloid DCs, and provided important preclinical data for rapid development of potent whole tumor lysate-loaded DC vaccines that are applicable to many tumor types.     

Incorporation of Porcine Adenovirus 4 Fiber Protein Enhances Infectivity of Adenovirus Vector on Dendritic Cells: Implications for Immune-Mediated Cancer Therapy

One strategy in cancer immunotherapy is to capitalize on the key immunoregulatory and antigen presenting capabilities of dendritic cells (DCs). This approach is dependent on efficient delivery of tumor specific antigens to DCs, which subsequently induce an anti-tumor T-cell mediated immune response. Human adenovirus serotype 5 (HAdV5) has been used in human studies for gene delivery, but has limited infection in DCs, which lack the proper receptors. Addition of the porcine fiber knob (PK) from porcine adenovirus type 4 to HAdV5 allows the virus to deliver genetic material via binding to glycosylated surface proteins and bypasses the coxsackie-and-adenovirus receptor required by wild-type HAdV5. In this study we explored the potential therapeutic applications of an adenovirus with PK-based tropism against cancers expressing mesothelin. Infectivity and gene transfer assays were used to compare Ad5-PK to wild-type HAdV5. Mouse models were used to demonstrate peptide specificity and T-cell responses. We show that the PK modification highly augmented infection of DCs, including the CD141+ DC subset, a key subset for activation of naïve CD8+ T-cells. We also show that Ad5-PK increases DC infectivity and tumor specific antigen expression. Finally, vaccination of mice with the Ad5-PK vector resulted in enhanced T-cell-mediated interferon gamma (IFN-γ) release in response to both mesothelin peptide and a tumor line expressing mesothelin. Ad5-PK is a promising tool for cancer immunotherapy as it improves infectivity, gene transfer, protein expression, and subsequent T-cell activation in DCs compared to wild-type HAdV5 viruses.