Human breast cancer-derived soluble factors facilitate CCL19-induced chemotaxis of human dendritic cells.

Breast cancer remains as a challenging disease with high mortality in women. Increasing evidence points the importance of understanding a crosstalk between breast cancers and immune cells, but little is known about the effect of breast cancer-derived factors on the migratory properties of dendritic cells (DCs) and their consequent capability in inducing T cell immune responses. Utilizing a unique 3D microfluidic device, we here showed that breast cancers (MCF-7, MDA-MB-231, MDA-MB-436 and SK-BR-3)-derived soluble factors increase the migration of DCs toward CCL19. The enhanced migration of DCs was mainly mediated via the highly activated JNK/c-Jun signaling pathway, increasing their directional persistence, while the velocity of DCs was not influenced, particularly when they were co-cultured with triple negative breast cancer cells (TNBCs or MDA-MB-231 and MDA-MB-436). The DCs up-regulated inflammatory cytokines IL-1ß and IL-6 and induced T cells more proliferative and resistant against activation-induced cell death (AICD), which secret high levels of inflammatory cytokines IL-1ß, IL-6 and IFN-γ. This study demonstrated new possible evasion strategy of TNBCs utilizing their soluble factors that exploit the directionality of DCs toward chemokine responses, leading to the building of inflammatory milieu which may support their own growth.

Effective Delivery of Antigen-Encapsulin Nanoparticle Fusions to Dendritic Cells Leads to Antigen-Specific Cytotoxic T Cell Activation and Tumor Rejection.

In cancer immunotherapy, robust and efficient activation of cytotoxic CD8(+) T cell immune responses is a promising, but challenging task. Dendritic cells (DCs) are well-known professional antigen presenting cells that initiate and regulate antigen-specific cytotoxic CD8(+) T cells that kill their target cells directly as well as secrete IFN-γ, a cytokine critical in tumor rejection. Here, we employed recently established protein cage nanoparticles, encapsulin (Encap), as antigenic peptide nanocarriers by genetically incorporating the OT-1 peptide of ovalbumin (OVA) protein to the three different positions of the Encap subunit. With them, we evaluated their efficacy in activating DC-mediated antigen-specific T cell cytotoxicity and consequent melanoma tumor rejection in vivo. DCs efficiently engulfed Encap and its variants (OT-1-Encaps), which carry antigenic peptides at different positions, and properly processed them within phagosomes. Delivered OT-1 peptides were effectively presented by DCs to naïve CD8(+) T cells successfully, resulting in the proliferation of antigen-specific cytotoxic CD8(+) T cells. OT-1-Encap vaccinations in B16-OVA melanoma tumor bearing mice effectively activated OT-1 peptide specific cytotoxic CD8(+) T cells before or even after tumor generation, resulting in significant suppression of tumor growth in prophylactic as well as therapeutic treatments. A large number of cytotoxic CD8(+) T cells that actively produce both intracellular and secretory IFN-γ were observed in tumor-infiltrating lymphocytes collected from B16-OVA tumor masses originally vaccinated with OT-1-Encap-C upon tumor challenges. The approaches we describe herein may provide opportunities to develop epitope-dependent vaccination systems that stimulate and/or modulate efficient and epitope-specific cytotoxic T cell immune responses in nonpathogenic diseases.

Intrinsic features of the CD8α(-) dendritic cell subset in inducing functional T follicular helper cells.

T follicular helper (Tfh) cells, a true B cell helper, have a critical role in enhancing humoral immune responses. However, the initial differentiation of Tfh cells by dendritic cells (DCs), the most potent antigen presenting cells, has not been clearly understood, particularly in the knowledge of the two major conventional dendritic cell subsets, CD8α(+) DCs or CD8α(-) DCs. Here we demonstrated that the localization of CD8α(-) DCs in the marginal zone (MZ) bridging channels is closely associated with the induction of CXCR5(+)CCR7(low) Tfh cells. We also showed that the major source of IL-6 for inducing Tfh cells is provided from the activated CD4(+) T cells induced by CD8α(-) DCs, and IL-6 directly secreted from the DC subsets seems minor. CD8α(-) DCs were superior in inducing functional Tfh cells over other antigen presenting cells including B cells. We here observed the unknown intrinsic features of the DC subsets, suggesting the potential of utilizing the CD8α(-) DC subset as therapeutic vaccine for the regulation of humoral immune responses.

CD8α(-) Dendritic Cells Induce Antigen-Specific T Follicular Helper Cells Generating Efficient Humoral Immune Responses.

Recent studies on T follicular helper (Tfh) cells have significantly advanced our understanding of T cell-dependent B cell responses. However, little is known about the early stage of Tfh cell commitment by dendritic cells (DCs), particularly by the conventional CD8α(+) and CD8α(-) DC subsets. We show that CD8α(-) DCs localized at the interfollicular zone play a pivotal role in the induction of antigen-specific Tfh cells by upregulating the expression of Icosl and Ox40l through the non-canonical NF-κB signaling pathway. Tfh cells induced by CD8α(-) DCs function as true B cell helpers, resulting in significantly increased humoral immune responses against various human pathogenic antigens, including Yersinia pestis LcrV, HIV Gag, and hepatitis B surface antigen. Our findings uncover a mechanistic role of CD8α(-) DCs in the initiation of Tfh cell differentiation and thereby provide a rationale for investigating CD8α(-) DCs in enhancing antigen-specific humoral immune responses for improving vaccines and therapeutics.

In vitro generation of functional dendritic cells differentiated from CD34 negative cells isolated from human umbilical cord blood.

Dendritic cells (DCs) are the most potent antigen-presenting cells that play a crucial role in the initiation of an immune response. As DC-based therapeutic applications is increasing, large-scale DC production is required for transplantation. Human umbilical cord blood (UCB) has been shown to contain a rare and precious population of hematopoietic stem cells (HSCs), which can give rise to DCs. The CD34 antigen has been widely used as a cell surface marker to identify HSCs. In this study, we used CD34 antibody to isolate CD34(+) and CD34(-) cells and compared the ability to differentiate into DCs. We used a two-step method combined with the magnetic bead sorting system to isolate CD34(+) and CD34(-) cells from human UCB. Analysis of cellular properties and functionality using a migration assay and T cell proliferation assay revealed no significant differences between CD34(+) cells and CD34(-) cells in their ability to generate DCs.

Lumazine synthase protein cage nanoparticles as antigen delivery nanoplatforms for dendritic cell-based vaccine development.

PURPOSE: Protein cages are promising nanoplatform candidates for efficient delivery systems due to their homogenous size and structure with high biocompatibility and biodegradability. In this study, we investigate the potential of lumazine synthase protein cage as an antigen delivery system to dendritic cells (DCs), which induce antigen-specific T cell proliferation. MATERIALS AND METHODS: Ovalbumin (OVA) peptides OT-1 (SIINFEKL) and OT-2 (ISQAVHAAHAEINEAGR) were genetically inserted to lumazine synthase and each protein cage was over-expressed in Escherichia coli as a soluble protein. The efficiency of antigen delivery and the resulting antigen-specific T cell proliferation by DCs was examined in vitro as well as in vivo. RESULTS: We successfully generated and characterized OVA peptides carrying lumazine synthase protein cages. The OT-1 and OT-2 peptides carried by lumazine synthases were efficiently delivered and processed by DCs in vitro as well as in vivo, and induced proliferation of OT-1-specific CD8(+)T cells and OT-2-specific CD4(+)T cells. CONCLUSION: Our data demonstrate the potential of lumazine synthase protein cage being used as a novel antigen delivery system for DC-based vaccine development in future clinical applications.

Polyvalent display of monosaccharides on ferritin protein cage nanoparticles for the recognition and binding of cell-surface lectins.

Carbohydrate-lectin interactions are important in many biological events. Endogenous cell-surface lectins are attractive markers for the recognition and targeting. Human ferritin protein cage nanoparticles (HFPCNs) are prepared as delivery nanoplatforms and two different types of monosaccharide derivatives; maleimido group terminated-mannopyranoside and galactopyranoside. Uniform and polyvalent displays of mannoses or galactoses on the surface of HFPCNs are achieved by using site-specific thiol-maleimide Michael-type addition. Mannose- or galactose-displaying HFPCNs recognize and tightly bind to DC-SIGN or ASGP-R lectins on the surface of the mammalian cells, DCEK or HepG2 cells.

Ferritin protein cage nanoparticles as versatile antigen delivery nanoplatforms for dendritic cell (DC)-based vaccine development.

We utilized ferritin protein cage nanoparticles (FPCN) as antigen delivery nanoplatforms for DC-based vaccine development and investigated DC-mediated antigen-specific immune responses. Antigenic peptides, OT-1 (SIINFEKL) or OT-2 (ISQAVHAAHAEINEAGR) which are derived from ovalbumin, were genetically introduced either onto the exterior surface or into the interior cavity of FPCN. FPCN carrying antigenic peptides (OT-1-FPCN and OT-2-FPCN) were effectively delivered to DCs and processed within endosomes. Delivered antigenic peptides, OT-1 or OT-2, to DCs successfully induced antigen-specific CD8(+) or CD4(+) T cell proliferations both in vitro and in vivo. Naïve mice immunized with OT-1-FPCN efficiently differentiated OT-1 specific CD8(+) T cells into functional effector cytotoxic T cells resulting in selective killing of antigen-specific target cells. Effective differentiation of proliferated OT-2 specific CD4(+) T cells into functional CD4(+) Th1 and Th2 cells was confirmed with the productions of IFN-γ/IL-2 and IL-10/IL-13 cytokines, respectively. FROM THE CLINICAL EDITOR: In this study, the authors utilized ferritin protein cage nanoparticles as antigen delivery nanoplatforms for dendritic cell-based vaccine development and investigated DC-mediated antigen-specific immune responses using strong model antigens derived from ovalbumin, suggesting potential future clinical applicability of this or similar techniques.

Lung dendritic cells induce migration of protective T cells to the gastrointestinal tract.

Developing efficacious vaccines against enteric diseases is a global challenge that requires a better understanding of cellular recruitment dynamics at the mucosal surfaces. The current paradigm of T cell homing to the gastrointestinal (GI) tract involves the induction of α4ß7 and CCR9 by Peyer's patch and mesenteric lymph node (MLN) dendritic cells (DCs) in a retinoic acid-dependent manner. This paradigm, however, cannot be reconciled with reports of GI T cell responses after intranasal (i.n.) delivery of antigens that do not directly target the GI lymphoid tissue. To explore alternative pathways of cellular migration, we have investigated the ability of DCs from mucosal and nonmucosal tissues to recruit lymphocytes to the GI tract. Unexpectedly, we found that lung DCs, like CD103(+) MLN DCs, up-regulate the gut-homing integrin α4ß7 in vitro and in vivo, and induce T cell migration to the GI tract in vivo. Consistent with a role for this pathway in generating mucosal immune responses, lung DC targeting by i.n. immunization induced protective immunity against enteric challenge with a highly pathogenic strain of Salmonella. The present report demonstrates novel functional evidence of mucosal cross talk mediated by DCs, which has the potential to inform the design of novel vaccines against mucosal pathogens.

TLR2 signaling in tubular epithelial cells regulates NK cell recruitment in kidney ischemia-reperfusion injury.

Damage-associated molecular patterns released from damaged kidney cells initiate postischemic inflammation, an essential step in the progression of kidney ischemia-reperfusion injury (IRI). However, the mechanism that coordinates this highly specific process in ischemic kidneys remains to be clarified. Previously, we demonstrated that CD137 from NK cells specifically stimulates CD137 ligand (CD137L) on tubular epithelial cells (TECs) such that TECs produced the high CXCR2 chemokine levels required for neutrophil chemotaxis. We report in the present study that endogenous TLR2 ligands released from ischemic TECs induce CCR5 chemokine expression, which is critical to promoting NK cell recruitment. By implanting CD137L(-/-) TECs into the kidney capsule of TLR2(-/-) mice, we further showed that TLR2-mediated NK cell recruitment is an uncoupled event that can occur independently of CD137L signaling in TECs, which is responsible for recruiting neutrophils. Therefore, our findings identify TECs as both a target for kidney damage and also as a master regulator that actively modulates stepwise signaling, leading to the initiation and amplification of acute sterile inflammation that inflicts kidney IRI. Being clinically important, the signaling pathway of innate receptors in epithelial cells may therefore be a good target to block acute sterile inflammation resulting from tissue damage, including kidney IRI.

Suppression of miRNA-708 by polycomb group promotes metastases by calcium-induced cell migration.

The progression of cancer to metastatic disease is a major cause of death. We identified miR-708 being transcriptionally repressed by polycomb repressor complex 2-induced H3K27 trimethylation in metastatic breast cancer. miR-708 targets the endoplasmic reticulum protein neuronatin to decrease intracellular calcium level, resulting in reduction of activation of ERK and FAK, decreased cell migration, and impaired metastases. Ectopic expression of neuronatin refractory to suppression by miR-708 rescued cell migration and metastasis defects. In patients with breast cancer, miR-708 expression was decreased in lymph node and distal metastases, suggesting a metastasis-suppressive role. Our findings uncover a mechanistic role for miR-708 in metastasis and provide a rationale for developing miR-708 as a therapeutic agent against metastatic breast cancer.

Three dimensional multicellular co-cultures and anti-cancer drug assays in rapid prototyped multilevel microfluidic devices.

This report presents a multilevel microfluidic platform for robust construction of hydrogel scaffold in microchannels and its application to three dimensional (3D) multicellular co-cultures and assays. A new rapid prototyping method based on soft lithography using multi-layered adhesive tapes is also introduced. We have successfully cultured MCF-7 breast cancer cell line more than 11 days with > 98 % viability, and co-cultured MDA-MB-231 breast cancer cells and NIH/3T3 fibroblasts in separate compartments. This multilevel microfluidic device with a cell-laden hydrogel microstructure has also been applied for anticancer drug assays in multicellular niches. Here we tested the effect of estrogen receptor (ER) antagonist drug, tamoxifen, on the growth of ER positive MCF-7 cells in microchannels. The inhibitory effect of tamoxifen on the growth of MCF-7 cells was diminished when they were co-cultured with ER negative MDA-MB-231 cells. The rapid prototyped multilevel microfluidic devices would provide simple, easy to use, low cost, robust, and reproducible cell-based assay platforms for potential end-users such as biologists and pharmacists.

Induction of pulmonary mucosal immune responses with a protein vaccine targeted to the DEC-205/CD205 receptor.

It is of great interest to develop a pneumonic plague vaccine that would induce combined humoral and cellular immunity in the lung. Here we investigate a novel approach based on targeting of dendritic cells using the DEC-205/CD205 receptor (DEC) via the intranasal route as way to improve mucosal cellular immunity to the vaccine. Intranasal administration of Yersinia pestis LcrV (V) protein fused to anti-DEC antibody together with poly IC as an adjuvant induced high frequencies of IFN-γ secreting CD4(+) T cells in the airway and lung as well as pulmonary IgG and IgA antibodies. Anti-DEC:LcrV was more efficient to induce IFN-γ/TNF-α/IL-2 secreting polyfunctional CD4(+) T cells when compared to non-targeted soluble protein vaccine. In addition, the intranasal route of immunization with anti-DEC:LcrV was associated with improved survival upon pulmonary challenge with the virulent CO92 Y. pestis. Taken together, these data indicate that targeting dendritic cells via the mucosal route is a potential new avenue for the development of a mucosal vaccine against pneumonic plague.

Targeting of LcrV virulence protein from Yersinia pestis to dendritic cells protects mice against pneumonic plague.

To help design needed new vaccines for pneumonic plague, we targeted the Yersinia pestis LcrV protein directly to CD8α(+) DEC-205(+) or CD8α(-) DCIR2(+) DC along with a clinically feasible adjuvant, poly IC. By studying Y. pestis in mice, we could evaluate the capacity of this targeting approach to protect against a human pathogen. The DEC-targeted LcrV induced polarized Th1 immunity, whereas DCIR2-targeted LcrV induced fewer CD4(+) T cells secreting IFN-γ, but higher IL-4, IL-5, IL-10, and IL-13 production. DCIR-2 targeting elicited higher anti-LcrV Ab titers than DEC targeting, which were comparable to a protein vaccine given in alhydrogel adjuvant, but the latter did not induce detectable T-cell immunity. When DEC- and DCIR2-targeted and F1-V+ alhydrogel-vaccinated mice were challenged 6 wk after vaccination with the virulent CO92 Y. pestis, the protection level and Ab titers induced by DCIR2 targeting were similar to those induced by F1-V protein with alhydrogel vaccination. Therefore, LcrV targeting to DC elicits combined humoral and cellular immunity, and for the first time with this approach, also induces protection in a mouse model for a human pathogen.

Improved cellular and humoral immune responses in vivo following targeting of HIV Gag to dendritic cells within human anti-human DEC205 monoclonal antibody.

Protein vaccines for T-cell immunity are not being prioritized because of poor immunogenicity. To overcome this hurdle, proteins are being targeted to maturing dendritic cells (DCs) within monoclonal antibodies (mAbs) to DC receptors. To extend the concept to humans, we immunized human immunoglobulin-expressing mice with human DEC205 (hDEC205) extracellular domain. 3D6 and 3G9 mAbs were selected for high-affinity binding to hDEC205. In addition, CD11c promoter hDEC205 transgenic mice were generated, and 3G9 was selectively targeted to DCs in these animals. When mAb heavy chain was engineered to express HIV Gag p24, the fusion mAb induced interferon-γ- and interleukin-2-producing CD4(+) T cells in hDEC205 transgenic mice, if polynocinic polycytidylic acid was coadministered as an adjuvant. The T-cell response was broad, recognizing at least 3 Gag peptides, and high titers of anti-human immunoglobulin G antibody were made. Anti-hDEC205 also improved the cross-presentation of Gag to primed CD8(+) T cells from HIV-infected individuals. In all tests, 3D6 and 3G9 targeting greatly enhanced immunization relative to nonbinding control mAb. These results provide preclinical evidence that in vivo hDEC205 targeting increases the efficiency with which proteins elicit specific immunity, setting the stage for proof-of-concept studies of these new protein vaccines in human subjects.

Identification of antigen-presenting dendritic cells in mouse aorta and cardiac valves.

Presumptive dendritic cells (DCs) bearing the CD11c integrin and other markers have previously been identified in normal mouse and human aorta. We used CD11c promoter-enhanced yellow fluorescent protein (EYFP) transgenic mice to visualize aortic DCs and study their antigen-presenting capacity. Stellate EYFP(+) cells were readily identified in the aorta and could be double labeled with antibodies to CD11c and antigen-presenting major histocompatability complex (MHC) II products. The DCs proved to be particularly abundant in the cardiac valves and aortic sinus. In all aortic locations, the CD11c(+) cells localized to the subintimal space with occasional processes probing the vascular lumen. Aortic DCs expressed little CD40 but expressed low levels of CD1d, CD80, and CD86. In studies of antigen presentation, DCs selected on the basis of EYFP expression or binding of anti-CD11c antibody were as effective as DCs similarly selected from the spleen. In particular, the aortic DCs could cross-present two different protein antigens on MHC class I to CD8(+) TCR transgenic T cells. In addition, after intravenous injection, aortic DCs could capture anti-CD11c antibody and cross-present ovalbumin to T cells. These results indicate that bona fide DCs are a constituent of the normal aorta and cardiac valves.

Dropout alignment allows homology recognition and evolutionary analysis of rDNA intergenic spacers.

Subrepeats within the ribosomal gene (rDNA) intergenic spacer (IGS) play an important role in enhancing RNA polymerase I transcription. Despite this functional role and presumed selective constraint, there is surprisingly little sequence similarity among IGS subrepeats of different species. This sequence dissimilarity corresponds with the fast insertion-deletion (indel) rates observed in short mononucleotide microsatellites (here referred to as poly[N] runs, where N is any nucleotide), which are relatively abundant in rDNA IGS subrepeats. Some species have different types of IGS subrepeats that share species-specific poly(N) run patterns. This finding indicates that many IGS subrepeats within species share a common evolutionary history. Furthermore, by aligning sequences after modifying them by the dropout method, i.e., by disregarding poly(N) runs during the sequence aligning step, we sought to uncover evolutionarily shared similarities that fail to be recognized by current alignment programs. To ensure that the improved similarities in the computed alignments are not a chance artifact, we calibrated and corrected the IGS subrepeat sequences for the influence of repeat length and estimated the statistical significance of the alignments (in terms of a stringent p-value) obtained by the dropout method by comparing them to null models constructed using random sequence sets from the same genomes. We found that most diverse kinds of rDNA IGS subrepeats in one species must have been derived from a common ancestral subrepeat, and that it is possible to infer the evolutionary relationships among the IGS subrepeats of different species by comparative genomics methods based on dropout alignments.

Broad T cell immunity to the LcrV virulence protein is induced by targeted delivery to DEC-205/CD205-positive mouse dendritic cells.

There is a need for a more efficient vaccine against the bacterium Yersinia pestis, the agent of pneumonic plague. The F1-LcrV (F1-V) subunit vaccine in alhydrogel is known to induce humoral immunity. In this study, we utilized DC to investigate cellular immunity. We genetically engineered the LcrV virulence protein into the anti-DEC-205/CD205 mAb and thereby targeted the conjugated protein directly to mouse DEC-205(+) DC in situ. We observed antigen-specific CD4(+) T cell immunity measured by intracellular staining for IFN-gamma in three different mouse strains (C57BL/6, BALB/c, and C3H/HeJ), while we could not observe such T cell responses with F1-V vaccine in alhydrogel. Using a peptide library for LcrV protein, we identified two or more distinct CD4(+) T cell mimetopes in each MHC haplotype, consistent with the induction of broad immunity. When compared to nontargeted standard protein vaccine, DC targeting greatly increased the efficiency for inducing IFN-gamma-producing T cells. The targeted LcrV protein induced antibody responses to a similar extent as the F1-V subunit vaccine, but Th1-dependent IgG2a and IgG2c isotypes were observed only after anti-DEC-205:LcrV mAb immunization. This study sets the stage for the analysis of functional roles of IFN-gamma-producing T cells in Y. pestis infection.

Generation and application of new rat monoclonal antibodies against synthetic FLAG and OLLAS tags for improved immunodetection.

Previously, we prepared monoclonal antibodies (mAbs) by immunizing rats with the recombinant fusion proteins of mouse Langerin/CD207, which contained a flexible linker sequence from E. coli OmpF and a FLAG epitope. We found many of new rat mAbs were not reactive to mouse Langerin, and here we identify the epitopes of two of these IgG mAbs, L2 and L5, and assess their efficacy in various immunodetection methods. MAb L5 is a rat IgG mAb against the FLAG epitope, which detected both N-terminal and C-terminal FLAG tagged protein 2 to 8 times better than the conventional anti-FLAG mAb M2 by Western blot. For mAb L2, we found its epitope to be a 14 amino acid sequence SGFANELGPRLMGK which consisted of both sequences from the OmpF derived linker and mouse Langerin. This epitope sequence was named OLLAS (E. coliOmpF Linker and mouse Langerin fusion Sequence), and mAb L2 as mAb OLLA-2. When the OLLAS sequence was inserted into recombinant proteins at N-terminal, C-terminal, or internal sites, the OLLAS tag was detected by mAb OLLA-2 with very high sensitivity compared to other conventional epitope tags and anti-tag mAbs. MAb OLLA-2 recognized OLLAS tagged proteins with at least 100-fold more sensitivity than anti-FLAG M2 and anti-V5 mAbs in Western blot analyses. We also find the OLLAS epitope to be superior in immunoprecipitation and other immunodetection methods, such as fluorescent immunohistochemistry and flow cytometry. In the process, we successfully utilized the OLLAS epitope sequence as an internal linker for fusion between the engineered mAb and the antigen, and thus achieved improved immunodetection.