Mediators of innate immunity that target immature, but not mature, dendritic cells induce antitumor immunity when genetically fused with nonimmunogenic tumor antigens.

Chemokine receptors are differentially expressed on immature and mature dendritic cells (DC). Herein, we demonstrate for the first time that murine antimicrobial peptides beta-defensins 2 and 3 bind murine CCR6, similarly to inflammatory chemokine macrophage-inflammatory protein 3alpha, and they chemoattract bone marrow-derived immature, but not mature DC. Using various chemokines or defensins fused with nonimmunogenic tumor Ags, we studied their capacity to delivery Ags to subsets of immune cells to elicit antitumor immunity. We demonstrate that DNA immunizations with fusion constructs with beta-defensin 2 or inflammatory chemokines that target immature DC, but not homeostatic chemokines secondary lymphoid tissue chemokine, CCL21, or stromal cell-derived factor 1, CXCL12, which chemoattract mature DC, elicit humoral, protective, and therapeutic immunity against two different syngeneic lymphomas.     

Tumor-produced secreted form of binding of immunoglobulin protein elicits antigen-specific tumor immunity

Binding of immunoglobulin protein (BiP) is a major molecular chaperone localized in endoplasmic reticulum (ER). It has been demonstrated to interact with nascent Ig. However, contrary to other ER-resident heat shock proteins such as gp96, calreticulin, and ORP150, it is not clear whether tumor-derived BiP plays a role in inducing antitumor immunity. In this study, we show that the tumor-derived secreted form of BiP is capable of inducing antitumor CD8(+) T cell responses. We constructed an ER-retention signal KDEL-deleted mutant of BiP cDNA and transfected it to tumor cells, which resulted in continuous secretion of tumor-derived BiP into the extracellular milieu. We show that this secreted BiP is taken up by bone marrow-derived dendritic cells, and thereafter BiP-associated Ag peptide is cross-presented in association with MHC class I molecules, resulting in elicitation of an Ag-specific CD8(+) T cell response and antitumor effect. This strategy to boost antitumor immune responses shows that a tumor could be its own cellular vaccine via gene modification of the secretion of the tumor Ag-BiP complex.     

Expression of a soluble TGF-beta receptor by tumor cells enhances dendritic cell/tumor fusion vaccine efficacy

Dendritic cell (DC)-based antitumor immunotherapy is a promising cancer therapy. We have previously shown that tumor-derived TGF-beta limits the efficacy of the DC/tumor fusion vaccine in mice. In the current study we investigated the effect of neutralizing tumor-derived TGF-beta on the efficacy of the DC/tumor fusion vaccine. An adenovirus encoding human TGF-beta receptor type II fused to the Fc region of human IgM (Adv-TGF-beta-R) or a control adenovirus encoding LacZ (Adv-LacZ) was used to express a soluble form of the neutralizing TGF-beta receptor (TGF-beta-R). Murine breast carcinoma cells, 4T1, but not bone marrow-derived DCs, were successfully transfected with Adv-TGF-beta-R (4T1+Adv-TGF-beta-R) using a multiplicity of infection of 300. Immunization with irradiated 4T1+Adv-TGF-beta-R tumor cells conferred enhanced antitumor immunity compared with immunization with irradiated 4T1+Adv-LacZ tumor cells. The DC/4T1+Adv-TGF-beta-R fusion vaccine offered enhanced protective and therapeutic efficacy compared with the DC/4T1-Adv-LacZ fusion vaccine. Because TGF-beta is known to induce regulatory T cells (Tregs), we further showed that the DC/4T1+Adv-TGF-beta-R fusion vaccine induced fewer CD4(+)CD25(+)Foxp3(+) Tregs than the DC/4T1+Adv-LacZ fusion vaccine in vitro and in vivo. The suppressive role of splenic CD4(+)CD25(+) Tregs isolated from mice immunized with DC/4T1+Adv-LacZ was demonstrated using a CTL killing assay. Similar enhanced therapeutic efficacy was observed in murine renal cell carcinoma, RenCa, which expresses a high level of TGF-beta. We conclude that the blockade of tumor-derived TGF-beta reduces Treg induction by the DC/tumor fusion vaccine and enhances antitumor immunity. This may be an effective strategy to enhance human DC-based antitumor vaccines.     

Activation of dendritic cells that cross-present tumor-derived antigen licenses CD8+ CTL to cause tumor eradication

The fate of naive CD8(+) T cells is determined by the environment in which they encounter MHC class I presented peptide Ags. The manner in which tumor Ags are presented is a longstanding matter of debate. Ag presentation might be mediated by tumor cells in tumor draining lymph nodes or via cross-presentation by professional APC. Either pathway is insufficient to elicit protective antitumor immunity. We now demonstrate using a syngeneic mouse tumor model, expressing an Ag derived from the early region 1A of human adenovirus type 5, that the inadequate nature of the antitumor CTL response is not due to direct Ag presentation by the tumor cells, but results from presentation of tumor-derived Ag by nonactivated CD11c(+) APC. Although this event results in division of naive CTL in tumor draining lymph nodes, it does not establish a productive immune response. Treatment of tumor-bearing mice with dendritic cell-stimulating agonistic anti-CD40 mAb resulted in systemic efflux of CTL with robust effector function capable to eradicate established tumors. For efficacy of anti-CD40 treatment, CD40 ligation of host APC is required because adoptive transfer of CD40-proficient tumor-specific TCR transgenic CTL into CD40-deficient tumor-bearing mice did not lead to productive antitumor immunity after CD40 triggering in vivo. CpG and detoxified LPS (MPL) acted similarly as agonistic anti-CD40 mAb with respect to CD8(+) CTL efflux and tumor eradication. Together these results indicate that dendritic cells, depending on their activation state, orchestrate the outcome of CTL-mediated immunity against tumors, leading either to an ineffective immune response or potent antitumor immunity.     

Prolonged antitumor NK cell reactivity elicited by CXCL10-expressing dendritic cells licensed by CD40L+ CD4+ memory T cells

Immunotherapy using dendritic cells (DCs) has the potential to activate both T cells and NK cells. We previously demonstrated the long-lasting antitumor responses by NK cells following immunization with bone marrow-derived DCs. In the current study, we demonstrate that long-term antitumor NK responses require endogenous DCs and a subset of effector memory CD4(+) T (CD4(+) T(EM)) cells. One month after DC immunization, injection of a tumor into DC-immunized mice leads to an increase in the expression of CXCL10 by endogenous DCs, thus directing NK cells into the white pulp where the endogenous DCs bridged CD4(+) T(EM) cells and NK cells. In this interaction, CD4(+) T(EM) cells express CD40L, which matures the endogenous DCs, and produce cytokines, such as IL-2, which activates NK cells. These findings suggest that DC vaccination can sustain long-term innate NK cell immunity but requires the participation of the adaptive immune system.     

Intralesional injection of adenovirus encoding CC chemokine ligand 16 inhibits mammary tumor growth and prevents metastatic-induced death after surgical removal of the treated primary tumor

The CC chemokine ligand (CCL)16 exerts chemotactic activity on human monocytes and lymphocytes. Although no murine homologous has been defined, the TSA mouse adenocarcinoma cells engineered to express human CCL16 are rapidly rejected by syngenic mice. An adenovirus encoding CCL16 (AdCCL16) was generated using a Cre-Lox-based system and was used to determine whether this chemokine might also block pre-existing tumors. Both recombinant and viral CCL16 showed in vitro chemotactic activity for murine CD4(+) and CD8(+) lymphocytes and dendritic cells (DC). AdCCL16, but not the control empty vector, when injected in established nodules significantly delayed tumor growth. Immunohistochemistry revealed accumulation of CD4(+) and CD8(+) T cells and DC in the treated tumors as well as in draining lymph nodes. DC from such lymph nodes stimulated IFN-gamma by a T cell clone specific for the known TSA tumor-associated Ag (TAA), suggesting the tumor origin of these cells. Lymphocytes from the same nodes showed specific CTL activity against TSA tumor cells and their immunodominant TAA peptide. Antitumor activity required CD4, CD8, and IFN-gamma production, as shown using subset-depleted and knockout mice. Despite the robust and rapid immune response triggered by intratumoral injection of AdCCL16, the lesions were not completely rejected; however, the same treatment given before surgical excision of primary lesions prevented metastatic spread and cured 63% of mice bearing the 4T1 mammary adenocarcinoma, which is perhaps the most compelling model of spontaneous metastasis.     

Local expression of secondary lymphoid tissue chemokine delivered by adeno-associated virus within the tumor bed stimulates strong anti-liver tumor immunity

Development of an effective antitumor immune response depends on the appropriate interaction of effector and target cells. Thus, the expression of chemokines within the tumor may induce a more potent antitumor immune response. Secondary lymphoid tissue chemokine (SLC) is known to play a critical role in establishing a functional microenvironment in secondary lymphoid tissues. Its capacity to attract dendritic cells (DCs) and colocalize them with T cells makes it a good therapeutic candidate against cancer. In this study, we used SLC as a treatment for tumors established from a murine hepatocellular carcinoma model. SLC was encoded by recombinant adeno-associated virus (rAAV), a system chosen for the low host immunity and high efficiency of transduction, enabling long-term expression of the gene of interest. As a result, rAAV-SLC induced a significant delay of tumor progression, which was paralleled by a profound infiltration of DCs and activated CD4(+) T cells and CD8(+) T cells (CD3(+) CD69(+) cells) into the tumor site. In addition, rAAV-SLC treatment was also found to reduce tumor growth in nude mice, most likely due to inhibition of neoangiogenesis. In conclusion, local expression of SLC by rAAV represents a promising approach to induce immune-mediated regression of malignant tumors.     

CD40 ligand promotes priming of fully potent antitumor CD4(+) T cells in draining lymph nodes in the presence of apoptotic tumor cells.

The presence or absence of CD4(+) T cell help can determine the direction of adaptive immune responses toward either cross-priming or cross-tolerance. It has been demonstrated that interactions of CD40-CD40 ligand can replace CD4(+) T cell help and enable dendritic cells to prime cytotoxic T cells. Here, we demonstrate that antitumor reactivity induced in regional lymph nodes (LNs) by s.c. injection of CD40 ligand (CD40L)-transduced tumor (MCA205 CD40L) showed far superior therapeutic efficacy against established brain tumors of a weakly immunogenic fibrosarcoma, MCA205, when adoptively transferred. Coinjection of apoptotic, but not necrotic parental tumor cells with CD40L-expressing tumor cells caused a strong synergistic induction of antitumor reactivity in tumor-draining LNs. Freshly isolated T cells from LNs immunized with apoptotic parental tumor cells and MCA205 CD40L were capable of mediating regression of the parental tumor in vivo. In contrast, T cells derived from LNs immunized without MCA205 CD40L required ex vivo anti-CD3/IL-2 activation to elicit therapeutic activity. On anti-CD3/IL-2 activation, cells from LNs immunized with MCA205 CD40L exhibited superior per cell antitumor reactivity. An in vitro depletion study revealed that either CD4(+) or CD8(+) T cells could mediate therapeutic efficacy but that the antitumor efficacy mediated by CD4(+) T cells was far superior. Cytosolic flow cytometric analyses indicated that priming of CD4(+) cells in LNs draining CD40L-expressing tumors was polarized to the Th1 type. This is the first report that fully potent antitumor CD4(+) T cell priming was promoted by s.c. injection of CD40L-transduced tumor in the presence of apoptotic tumor cells.     

EBV-induced molecule 1 ligand chemokine (ELC/CCL19) promotes IFN-gamma-dependent antitumor responses in a lung cancer model

The antitumor efficacy of EBV-induced molecule 1 ligand CC chemokine (ELC/CCL19) was evaluated in a murine lung cancer model. The ability of ELC/CCL19 to chemoattract both dendritic cells and T lymphocytes formed the rationale for this study. Compared with diluent-treated tumor-bearing mice, intratumoral injection of recombinant ELC/CCL19 led to significant systemic reduction in tumor volumes (p < 0.01). ELC/CCL19-treated mice exhibited an increased influx of CD4 and CD8 T cell subsets as well as dendritic cells at the tumor sites. These cell infiltrates were accompanied by increases in IFN-gamma, MIG/CXCL9, IP-10/CXCL10, GM-CSF, and IL-12 but a concomitant decrease in the immunosuppressive molecules PGE(2) and TGFbeta. Transfer of T lymphocytes from ELC/CCL19 treated tumor-bearing mice conferred the antitumor therapeutic efficacy of ELC/CCL19 to naive mice. ELC/CCL19 treated tumor-bearing mice showed enhanced frequency of tumor specific T lymphocytes secreting IFN-gamma. In vivo depletion of IFN-gamma, MIG/CXCL9, or IP-10/CXCL10 significantly reduced the antitumor efficacy of ELC/CCL19. These findings provide a strong rationale for further evaluation of ELC/CCL19 in tumor immunity and its use in cancer immunotherapy.     

Distinct functional capacities of mouse thymic and splenic dendritic cell populations

Dendritic cells (DC) are antigen-presenting cells that activate naive T cells. Murine DC are a heterogeneous population and can be subdivided into distinct subsets with different immune regulatory functions, namely the conventional DC (cDC), which include the CD8(+)Sirpalpha(-) and CD8(-)Sirpalpha(+) DC, and the plasmacytoid DC (pDC). In this study, the phenotype and function of DC subsets in both the thymus and spleen were compared. Significant differences between the thymic and splenic DC were observed in the expression of genes encoding chemokine receptors (CCRs), toll-like receptors (TLRs) and chemokines. Thymic DC expressed high levels of genes encoding a unique set of chemokines (CCL17 and CCL22) known to be important for T-cell development. Moreover, the capacity of the DC from the two organs to produce IL-6, IFN-alpha and IL-12p70 in response to the TLR 9 agonist CpG differed markedly, indicating intrinsic functional differences between subsets with similar surface phenotype. These results indicate that the microenvironment is an important factor that contributes to the functional specification of DC subsets in different lymphoid tissues.     

Effective treatment of inflammatory disease models with exosomes derived from dendritic cells genetically modified to express IL-4

In this study, we demonstrate that genetically modified bone marrow-derived dendritic cells (DC) and exosomes derived from the DC, expressing either secreted IL-4 or membrane-bound IL-4, can reduce the severity and the incidence of established collagen-induced arthritis and inhibit inflammation of delayed-type hypersensitivity (DTH) in mice. The ability of the DC and DC-derived exosomes to suppress the DTH response was MHC class II and, in part, Fas ligand/Fas dependent. The DC-derived exosomes were internalized by CD11c(+) DC in the dermis at the site of injection and in the draining lymph node as well as by CD11c(+) DC and F4/80(+) macrophages in the spleen. Moreover, adoptive transfer of CD11c(+) or CD3(+) splenic cells from mice treated with exosomes showed significant reduction of footpad swelling in the DTH model. These results demonstrate that administration of DC/IL-4 or exosomes derived from DC/IL-4 are able to modulate the activity of APC and T cells in vivo through a MHC class II and partly Fas ligand/Fas-dependent mechanism, resulting in effective treatment of established collagen-induced arthritis and suppression of the DTH inflammatory response. Thus, APC-derived exosomes could be used therapeutically for the treatment of autoimmune disease and inflammatory disorders.     

Tumor-derived exosomes are a source of shared tumor rejection antigens for CTL cross-priming

The initiation of T-cell-mediated antitumor immune responses requires the uptake and processing of tumor antigens by dendritic cells and their presentation on MHC-I molecules. Here we show in a human in vitro model system that exosomes, a population of small membrane vesicles secreted by living tumor cells, contain and transfer tumor antigens to dendritic cells. After mouse tumor exosome uptake, dendritic cells induce potent CD8+ T-cell-dependent antitumor effects on syngeneic and allogeneic established mouse tumors. Therefore, exosomes represent a novel source of tumor-rejection antigens for T-cell cross priming, relevant for immunointerventions.     

Exosomes as potent cell-free peptide-based vaccine. I. Dendritic cell-derived exosomes transfer functional MHC class I/peptide complexes to dendritic cells

Current immunization protocols in cancer patients involve CTL-defined tumor peptides. Mature dendritic cells (DC) are the most potent APCs for the priming of naive CD8(+) T cells, eventually leading to tumor eradication. Because DC can secrete MHC class I-bearing exosomes, we addressed whether exosomes pulsed with synthetic peptides could subserve the DC function consisting in MHC class I-restricted, peptide-specific CTL priming in vitro and in vivo. The priming of CTL restricted by HLA-A2 molecules and specific for melanoma peptides was performed: 1) using in vitro stimulations of total blood lymphocytes with autologous DC pulsed with GMP-manufactured autologous exosomes in a series of normal volunteers; 2) in HLA-A2 transgenic mice (HHD2) using exosomes harboring functional HLA-A2/Mart1 peptide complexes. In this study, we show that: 1). DC release abundant MHC class I/peptide complexes transferred within exosomes to other naive DC for efficient CD8(+) T cell priming in vitro; 2). exosomes require nature's adjuvants (mature DC) to efficiently promote the differentiation of melanoma-specific effector T lymphocytes producing IFN-gamma (Tc1) effector lymphocytes in HLA-A2 transgenic mice (HHD2). These data imply that exosomes might be a transfer mechanism of functional MHC class I/peptide complexes to DC for efficient CTL activation in vivo.     

CD16+ monocyte-derived macrophages activate resting T cells for HIV infection by producing CCR3 and CCR4 ligands

The CD16(+) monocyte (Mo) subset produces proinflammatory cytokines and is expanded in peripheral blood during progression to AIDS, but its contribution to HIV pathogenesis is unclear. In this study, we investigate the capacity of human CD16(+) and CD16(-) Mo subsets to render resting CD4(+) T cells permissive for HIV replication. We demonstrate that CD16(+) Mo preferentially differentiate into macrophages (Mphi) that activate resting T cells for productive HIV infection by producing the CCR3 and CCR4 ligands CCL24, CCL2, CCL22, and CCL17. CD16(+), but not CD16(-), Mo-derived Mphi from HIV-infected and -uninfected individuals constitutively produce CCL24 and CCL2. Furthermore, these chemokines stimulate HIV replication in CD16(-) Mo:T cell cocultures. Engagement of CCR3 and CCR4 by CCL24 and CCL2, respectively, along with stimulation via CD3/CD28, renders T cells highly permissive for productive HIV infection. Moreover, HIV replicates preferentially in CCR3(+) and CCR4(+) T cells. These findings reveal a new pathway of T cell costimulation for increased susceptibility to HIV infection via engagement of CCR3 and CCR4 by chemokines constitutively produced by CD16(+) Mo/Mphi. Thus, expansion of CD16(+) Mo in peripheral blood of HIV-infected patients and their subsequent recruitment into tissues may contribute to chronic immune activation and establishment of viral reservoirs in resting T cells.     

Diverse and potent chemokine production by lung CD11bhigh dendritic cells in homeostasis and in allergic lung inflammation

Lung CD11c(high) dendritic cells (DC) are comprised of two major phenotypically distinct populations, the CD11b(high) DC and the integrin alpha(E)beta(7)(+) DC (CD103(+) DC). To examine whether they are functionally distinguishable, global microarray studies and real-time PCR analysis were performed. Significant differences between the two major CD11c(high) DC types in chemokine mRNA expression were found. CD11b(high) DC is a major secretory cell type and highly expressed at least 16 chemokine mRNA in the homeostatic state, whereas CD103(+) DC highly expressed only 6. Intracellular chemokine staining of CD11c(high) lung cells including macrophages, and ELISA determination of sort-purified CD11c(high) cell culture supernatants, further showed that CD11b(high) DC produced the highest levels of 9 of 14 and 5 of 7 chemokines studied, respectively. Upon LPS stimulation in vitro and in vivo, CD11b(high) DC remained the highest producer of 7 of 10 of the most highly produced chemokines. Induction of airway hyperreactivity and lung inflammation increased lung CD11b(high) DC numbers markedly, and they produced comparable or higher amounts of 11 of 12 major chemokines when compared with macrophages. Although not a major producer, CD103(+) DC produced the highest amounts of the Th2-stimulating chemokines CCL17/thymus and activation-related chemokine and CCL22/monocyte-derived chemokine in both homeostasis and inflammation. Significantly, CCL22/monocyte-derived chemokine exhibited regulatory effects on CD4(+) T cell proliferation. Further functional analysis showed that both DC types induced comparable Th subset development. These studies showed that lung CD11b(high) DC is one of the most important leukocyte types in chemokine production and it is readily distinguishable from CD103(+) DC in this secretory function.     

NK and CD4 cells collaborate to protect against melanoma tumor formation in the brain

NK cells represent a potent immune effector cell type that have the ability to recognize and lyse tumors. However, the existence and function of NK cells in the traditionally "immune-privileged" CNS is controversial. Furthermore, the cellular interactions involved in NK cell anti-CNS tumor immunity are even less well understood. We administered non-Ag-loaded, immature dendritic cells (DC) to CD8alpha knockout (KO) mice and studied their anti-CNS tumor immune responses. DC administration induced dramatic antitumor immune protection in CD8alpha KO mice that were challenged with B16 melanoma both s.c. and in the brain. The CNS antitumor immunity was dependent on both CD4+ T cells and NK cells. Administration of non-Ag-loaded, immature DC resulted in significant CD4+ T cell and NK cell expansion in the draining lymph nodes at 6 days postvaccination, which persisted for 2 wk. Finally, DC administration in CD8alpha KO mice was associated with robust infiltration of CD4+ T cells and NK cells into the brain tumor parenchyma. These results represent the first demonstration of a potent innate antitumor immune response against CNS tumors in the absence of toxicity. Thus, non-Ag-loaded, immature DC administration, in the setting of CD8 genetically deficient mice, can induce dramatic antitumor immune responses within the CNS that surpass the effects observed in wild-type mice. Our results suggest that a better understanding of the cross-talk between DC and innate immune cells may provide improved methods to vaccinate patients with tumors located both systemically and within the CNS.     

Immune mechanism of the antitumor effects generated by bortezomib

Bortezomib, a proteasome inhibitor, is a chemotherapeutic drug that is commonly used to treat a variety of human cancers. The antitumor effects of bortezomib-induced tumor cell immunogenicity have not been fully delineated. In this study, we examined the generation of immune-mediated antitumor effects in response to treatment by bortezomib in a murine ovarian tumor model. We observed that tumor-bearing mice that were treated with bortezomib had CD8(+) T cell-mediated inhibition of tumor growth. Furthermore, the comparison of tumor cell-based vaccines that were produced from tumor cells treated or untreated with bortezomib showed vaccination with drug-treated tumor cell-based vaccines elicited potent tumor-specific CD8(+) T cell immune response with improved therapeutic antitumor effect in tumor-bearing mice. Conversely, the untreated tumor cell-based vaccines led to no appreciable antitumor response. Treatment of tumor cells with bortezomib led to the upregulation of Hsp60 and Hsp90 on the cell surface and promoted their phagocytosis by dendritic cells (DCs). However, cell surface expression of Hsp60, instead of Hsp90, is the more important determinant of whether bortezomib-treated tumor cells can generate tumor-specific CD8(+) T cells. CD11c(+) DCs that were treated with bortezomib in vitro had enhanced phagocytic activities. In addition, CD11c(+) DCs from bortezomib-treated tumor-bearing mice had increased maturation. At lower concentrations, bortezomib had no inhibitory effects on T cell proliferation. Taken together, our data indicate that bortezomib can render tumor cells immunogenic by upregulating the cell surface expression of heat shock protein 60 and heat shock protein 90, as well as improve DC function, which results in potent immune-mediated antitumor effects.     

Tumor-derived exosomes confer antigen-specific immunosuppression in a murine delayed-type hypersensitivity model

Exosomes are endosome-derived small membrane vesicles that are secreted by most cell types including tumor cells. Tumor-derived exosomes usually contain tumor antigens and have been used as a source of tumor antigens to stimulate anti-tumor immune responses. However, many reports also suggest that tumor-derived exosomes can facilitate tumor immune evasion through different mechanisms, most of which are antigen-independent. In the present study we used a mouse model of delayed-type hypersensitivity (DTH) and demonstrated that local administration of tumor-derived exosomes carrying the model antigen chicken ovalbumin (OVA) resulted in the suppression of DTH response in an antigen-specific manner. Analysis of exosome trafficking demonstrated that following local injection, tumor-derived exosomes were internalized by CD11c+ cells and transported to the draining LN. Exosome-mediated DTH suppression is associated with increased mRNA levels of TGF-β1 and IL-4 in the draining LN. The tumor-derived exosomes examined were also found to inhibit DC maturation. Taken together, our results suggest a role for tumor-derived exosomes in inducing tumor antigen-specific immunosuppression, possibly by modulating the function of APCs.     

Enhanced tumor responses to dendritic cells in the absence of CD8-positive cells

Wild-type mice immunized with MART-1 melanoma Ag-engineered dendritic cells (DC) generate strong Ag-specific immunity that has an absolute requirement for both CD8(+) and CD4(+) T cells. DC administration to CD8 alpha knockout mice displayed unexpectedly enhanced levels of protection to tumor challenge despite this deficiency in CD8(+) T cells and the inability to mount MHC class I-restricted immune responses. This model has the following features: 1) antitumor protection is Ag independent; 2) had an absolute requirement for CD4(+) and NK1.1(+) cells; 3) CD4(+) splenocytes are responsible for cytokine production; 4) lytic cells in microcytotoxicity assays express NK, but lack T cell markers (NK1.1(+) alpha beta TCR(-) CD3(-)); and 5) the lytic phenotype can be transferred to naive CD8 alpha knockout mice by NK1.1(+) splenocytes. Elucidation of the signaling events that activate these effective cytotoxic cells and the putative suppressive mechanisms in a wild-type environment may provide means to enhance the clinical activity of DC-based approaches.     

Vaccination with vascular progenitor cells derived from induced pluripotent stem cells elicits antitumor immunity targeting vascular and tumor cells

Vaccination of BALB/c mice with dendritic cells (DCs) loaded with the lysate of induced vascular progenitor (iVP) cells derived from murine-induced pluripotent stem (iPS) cells significantly suppressed the tumor of CMS-4 fibrosarcomas and prolonged the survival of CMS-4-inoculated mice. This prophylactic antitumor activity was more potent than that of immunization with DCs loaded with iPS cells or CMS-4 tumor cells. Tumors developed slowly in mice vaccinated with DCs loaded with iVP cells (DC/iVP) and exhibited a limited vascular bed. Immunohistochemistry and a tomato-lectin perfusion study demonstrated that the tumors that developed in the iVP-immunized mice showed a marked decrease in tumor vasculature. Immunization with DC/iVP induced a potent suppressive effect on vascular-rich CMS-4 tumors, a weaker effect on BNL tumors with moderate vasculature, and nearly no effect on C26 tumors with poor vasculature. Treatment of DC/iVP-immunized mice with a monoclonal antibody against CD4 or CD8, but not anti-asialo GM1, inhibited the antitumor activity. CD8⁺ T cells from DC/iVP-vaccinated mice showed significant cytotoxic activity against murine endothelial cells and CMS-4 cells, whereas CD8⁺ T cells from DC/iPS-vaccinated mice did not. DNA microarray analysis showed that the products of 29 vasculature-associated genes shared between genes upregulated by differentiation from iPS cells into iVP cells and genes shared by iVP cells and isolated Flk-1⁺ vascular cells in CMS-4 tumor tissue might be possible targets in the immune response. These results suggest that iVP cells from iPS cells could be used as a cancer vaccine targeting tumor vascular cells and tumor cells.