Expression of aberrantly glycosylated tumor mucin-1 on human DC after transduction with a fiber-modified adenoviral vector

BACKGROUND: DC-presenting tumor Ag are currently being developed to be used as a vaccine in human cancer immunotherapy. To increase chances for successful therapy it is important to deliver full-length tumor Ag instead of loading single peptides. METHODS: In this study we used a fiber-modified adenoviral vector (rAd5F35) containing full-length tumor Ag cDNA to transduce human monocyte (Mo)-derived DC in vitro. Cells were efficiently transduced and survived for at least 3 days after adenoviral transduction. Phenotype and function after maturation of Mo-DC were not impaired by infection with adenovirus particles. Expression of the tumor-associated Ag mucin-1 (MUC1) was detected using MAb defining different MUC1 glycoforms. RESULTS: Non-transduced mature Mo-DC express endogenous MUC1 with normal glycosylation. After transduction with the rAd5F35-MUC1 adenoviral vector, Mo-DC also expressed MUC1 with tumor-associated glycosylation (Tn and T glycoforms), although no changes in mRNA levels of relevant glycosyltransferases could be demonstrated. DISCUSSION: The presence of aberrantly glycosylated MUC1 may influence Ag presentation of the tumor glycoforms of MUC1 to immune cells, affecting tumor cell killing. These findings could be highly relevant to developing strategies for cancer immunotherapy based on DC vaccines using MUC1 as tumor Ag.     

Highly efficient transduction of human monocyte-derived dendritic cells with subgroup B fiber-modified adenovirus vectors enhances transgene-encoded antigen presentation to cytotoxic T cells

The efficiency of dendritic cells (DC) as immunotherapeutic vaccines critically depends on optimal delivery of target Ags. Although DC modified by subgroup C type 5 recombinant adenoviruses (rAd5) provide encouraging results, their clinical application is hampered by the need for high viral titers to achieve sufficient gene transfer, due to the lack of the Ad5 fiber receptor. We now demonstrate that rAd5 carrying subgroup B Ad fibers are up to 100-fold more potent than classical rAd5 for gene transfer and expression in human DC, rAd5 with a type 35 fiber (rAd5F35) being the most efficient vector. This improvement relates to a greater and faster virus entry and to an increased transgene expression especially following DC maturation. Furthermore, these new vectors possess enhanced synergistic effects with other activation signals to trigger DC maturation. Consequently, rAd5F35-infected DC engineered to express the gp100 melanoma-associated Ag largely exceed rAd5-infected DC in activating gp100-specific CTL. Finally, the DC infection pattern of rAd5F35 is fully conserved when DC are in the vicinity of primary skin-derived fibroblasts, suggesting this vector as a candidate for in vivo targeting of DC. Thus, subgroup B fiber-modified rAd5 constitute a major breakthrough in the exploitation of ex vivo rAd-targeted DC as clinically relevant vaccines and may also be suitable for in vivo genetic modification of DC.     

Intradermal delivery of adenoviral type-35 vectors leads to high efficiency transduction of mature, CD8+ T cell-stimulating skin-emigrated dendritic cells

Recombinant adenovirus (Ad) type 35 (rAd35) shows great promise as vaccine carrier with the advantage of low pre-existing immunity in human populations, in contrast to the more commonly used rAd5 vector. The rAd35 vector uses CD46 as a high-affinity receptor, which, unlike the rAd5 receptor, is expressed on human dendritic cells (DC), the most powerful APCs identified to date. In this study, we show that in contrast to rAd5, rAd35 infects migrated and mature CD83+ cutaneous DC with high efficiency (up to 80%), when delivered intradermally in an established human skin explant model. The high transduction efficiency is in line with high expression levels of CD46 detected on migratory cutaneous DC, which proved to be further increased upon intradermal administration of GM-CSF and IL-4. As compared with Ad5, these Ad35 infection characteristics translate into higher absolute numbers of skin-emigrated DC per explant that both express the transgene and are phenotypically mature. Finally, we demonstrate that upon intracutaneous delivery of a rAd35 vaccine encoding the circumsporozoite (CS) protein of Plasmodium falciparum, emigrated DC functionally express and process CS-derived epitopes and are capable of activating specific CD8+ effector T cells, as evidenced by activation of an HLA-A2-restricted CS-specific CD8+ T cell clone. Collectively, these data demonstrate the utility of rAd35 vectors for efficient in vivo human DC transduction.     

Myeloid and plasmacytoid dendritic cells are susceptible to recombinant adenovirus vectors and stimulate polyfunctional memory T cell responses

Although replication-incompetent recombinant adenovirus (rAd) type 5 is a potent vaccine vector for stimulating T and B cell responses, high seroprevalence of adenovirus type 5 (Ad5) within human populations may limit its clinical utility. Therefore, alternative adenovirus serotypes have been studied as vaccine vectors. In this study, we characterized the ability of rAd5 and rAd35 to infect and induce maturation of human CD11c(+) myeloid dendritic cells (MDCs) and CD123(+) plasmacytoid dendritic cells (PDCs), and their ability to stimulate Ag-specific T cells. Both MDCs and PDCs were found to express the primary receptor for Ad35 (CD46) but not Ad5 (coxsackie-adenovirus receptor; CAR). Both dendritic cell (DC) subsets were also more susceptible to rAd35 than to rAd5. MDCs were more susceptible to both rAd35 and rAd5 than were PDCs. Whereas rAd35 used CD46 for entry into DCs, entry of rAd5 may be through a CAR-independent pathway. Exposure to rAd35 but not rAd5 induced high levels of IFN-alpha in PDCs and phenotypic differentiation in both DC subsets. MDCs and PDCs exposed to either rAd5 or rAd35 encoding for CMV pp65 were able to present pp65 and activate CMV-specific memory CD8(+) and CD4(+) T cells in a dose-dependent manner, but MDCs stimulated the highest frequencies of pp65-specific T cells. Responding T cells expressed multiple functions including degranulation (CD107a surface mobilization) and production of IFN-gamma, IL-2, TNF-alpha, and MIP-1beta. Thus, the ability of rAd35 to naturally target important DC subsets, induce their maturation, and appropriately present Ag to T cells may herald greater in vivo immunogenicity than has been observed with rAd5.     

Transduction of human dendritic cells with a recombinant modified vaccinia Ankara virus encoding MUC1 and IL-2

The epithelial mucin MUC1 is considered an opportune target antigen for cancer immunotherapy, as it is over-expressed and exhibits aberrant glycosylation in malignant cells. Because dendritic cells (DC) are powerful initiators of immune responses, efforts have focused on tumor antigen-bearing DC as potent cancer vaccines. In this study we have characterized the transduction of monocyte-derived DC with a highly attenuated vaccinia virus vector [modified vaccinia Ankara (MVA)] encoding human MUC1 and the immunostimulatory cytokine IL-2. Analysis of transduced DC cultures generated from a number of donors revealed MUC1 expression in the range of 27-54% of the cells and a co-regulated secretion of bioactive IL-2. As shown by FACS analysis with MUCI-specific antibodies, the MVA-MUC1/IL-2-transduced DC predominantly expressed the fully processed glycoform of MUC1, typical of that displayed by normal epithelia. Over a 3-day period after transduction, transgene expression declined concurrent with an increase in MVA-induced cytopathic effects. The transduced DC stimulated allogeneic lymphocyte proliferation, indicating that DC immunostimulatory function is not impaired by vector transduction. In the presence of IL-2, MVA-transduced DC were able to enhance autologous lymphocyte proliferation. Also, vector expression was analyzed in DC cultures treated with TNF-alpha, a known DC maturation factor. As indicated by the up-regulation of several DC maturation markers, neither virus infection nor transgene expression influenced the maturation capacity of the cells. The MVA-MUC1/IL-2 vector effectively transduced both immature and TNF-alpha-matured DC. Overall, our results are encouraging for the clinical application of MVA-MUC1/IL-2-transduced DC.     

Increased antitumor capability of fiber-modified adenoviral vector armed with TRAIL against bladder cancers

Adenoviral vectors are widely used for cancer therapy and show a tumor-suppressing effect. However, bladder cancers are found to be resistant against infection of Ad5-derived adenoviral vector, limiting the application of the existing strategy of gene therapy. Therefore, efforts to develop novel types of adenoviral vector aimed for improving the viral infection and enhancing expression level of tumor-inhibiting transgene is urgently required. We constructed a 5/35 fiber-modified E1A-deleted adenoviral vector armed with TRAIL gene. Its ability to express this gene for inhibition of bladder cancer cell growth was investigated in our work. The results showed that this modification in fiber region facilitates adenoviral infection to bladder cancer, perhaps due to high expression of CD46 on target cell surface. Subsequently, we found an enhanced expression level of TRAIL mediated by 5/35 fiber-modified adenoviral vectors in bladder cancer cells, leading to an increased tumor-inhibiting capability of 5/35 adenoviral vector against bladder cancer cells. Consistently, growth of xenograft tumors in mice was also effectively inhibited by 5/35 fiber-modified vector-mediated gene therapy strategy. The 5/35 fiber-modified adenoviral vector-based gene transfer shows an improved efficacy against bladder cancers. The application of this novel gene therapy vector may benefit the patients in clinical bladder cancer treatment.     

Maturation of dendritic cells accompanies high-efficiency gene transfer by a CD40-targeted adenoviral vector.

Important therapeutic applications of genetically modified dendritic cells (DC) have been proposed; however, current vector systems have demonstrated only limited gene delivery efficacy to this cell type. By means of bispecific Abs, we have dramatically enhanced gene transfer to monocyte derived DC (MDDC) by retargeting adenoviral (Ad) vectors to a marker expressed on DC, CD40. Adenovirus targeted to CD40 demonstrated dramatic improvements in gene transfer relative to untargeted Ad vectors. Fundamental to the novelty of this system is the capacity of the vector itself to modulate the immunological status of the MDDC. This vector induces DC maturation as demonstrated phenotypically by increased expression of CD83, MHC, and costimulatory molecules, as well as functionally by production of IL-12 and an enhanced allostimulatory capacity in a MLR. In comparing this vector to other Ad-based gene transfer systems, we have illustrated that the features of DC maturation are not a function of the Ad particle, but rather a consequence of targeting to the CD40 marker. This vector approach may thus mediate not only high-efficiency gene delivery but also serve a proactive role in DC activation that could ultimately strengthen the utility of this vector for immunotherapy strategies.     

Dendritic cells induce MUC1 expression and polarization on human T cells by an IL-7-dependent mechanism

The MUC1 transmembrane mucin is expressed on the surface of activated human T cells; however, the physiologic signals responsible for the regulation of MUC1 in T cells are not known. The present studies demonstrate that IL-7, but not IL-2 or IL-4, markedly induces MUC1 expression on CD3+ T cells. MUC1 was also up-regulated by IL-15, but to a lesser extent than that found with IL-7. The results show that IL-7 up-regulates MUC1 on CD4+, CD8+, CD25+, CD69+, naive CD45RA+, and memory CD45RO+ T cells. In concert with induction of MUC1 expression by IL-7, activated dendritic cells (DC) that produce IL-7 up-regulate MUC1 on allogeneic CD3+ T cells. DC also induce MUC1 expression on autologous CD3+ T cells in the presence of recall Ag. Moreover, DC-induced MUC1 expression on T cells is blocked by a neutralizing anti-IL-7 Ab. The results also demonstrate that DC induce polarization of MUC1 on T cells at sites opposing the DC-T cell synapse. These findings indicate that DC-mediated activation of Ag-specific T cells is associated with induction and polarization of MUC1 expression by an IL-7-dependent mechanism.     

Human dendritic cells very efficiently present a heterologous antigen expressed on the surface of recombinant gram-positive bacteria to CD4+ T lymphocytes.

Recombinant Streptococcus gordonii expressing on the surface the C-fragment of tetanus toxin was tested as an Ag delivery system for human monocyte-derived dendritic cells (DCs). DCs incubated with recombinant S. gordonii were much more efficient than DCs pulsed with soluble C-fragment of tetanus toxin at stimulating specific CD4+ T cells as determined by cell proliferation and IFN-gamma release. Compared with DCs treated with soluble Ag, DCs fed with recombinant bacteria required 102- to 103-fold less Ag and were at least 102 times more effective on a per-cell basis for activating specific T cells. S. gordonii was internalized in DCs by conventional phagocytosis, and cytochalasin D inhibited presentation of bacteria-associated Ag, but not of soluble Ag, suggesting that phagocytosis was required for proper delivery of recombinant Ag. Bacteria were also very potent inducers of DC maturation, although they enhanced the capacity of DCs to activate specific CD4+ T cells at concentrations that did not stimulate DC maturation. In particular, S. gordonii dose-dependently up-regulated expression of membrane molecules (MHC I and II, CD80, CD86, CD54, CD40, CD83) and reduced both phagocytic and endocytic activities. Furthermore, bacteria promoted in a dose-dependent manner DC release of cytokines (IL-6, TNF-alpha, IL-1beta, IL-12, TGF-beta, and IL-10) and of the chemokines IL-8, RANTES, IFN-gamma-inducible protein-10, and monokine induced by IFN-gamma. Thus, recombinant Gram-positive bacteria appear a powerful tool for vaccine design due to their extremely high capacity to deliver Ags into DCs, as well as induce DC maturation and secretion of T cell chemoattractans.     

Adenoviruses activate human dendritic cells without polarization toward a T-helper type 1-inducing subset

Human monocyte-derived dendritic cells (DC) infected with recombinant adenoviruses (rAd) are promising candidate vaccines for inducing protective immunity against pathogens and tumors. However, since some viruses are known to negatively affect DC function, it is important to investigate the interactions between rAd and DC. We now show that infection by rAd enhances the immunostimulatory capacity of immature human monocyte-derived DC through the upregulation of the costimulatory molecules CD80, CD86, and CD40 and the major histocompatibility complex class I and II molecules. Although rAd infection fails to induce the secretion of interleukin-12 (IL-12) and only marginally induces the expression of the DC maturation marker CD83, it acts in synergy with CD40 triggering in rendering DC fully mature. rAd-infected DC triggered through CD40 produce more IL-12 and are more efficient in eliciting T-helper type 1 responses than DC activated by CD40 triggering only. rAd lacking one or more of the early regions, E1, E2A, E3, and E4, which play an important role in virus-host cell interactions are equally capable of DC activation. Efficient DC infection requires a high multiplicity of infection (>1,000), a fact which can be attributed to the absence of the coxsackievirus and adenovirus receptor on this cell type. Despite the poor ability of DC to be infected by rAd, which may be improved by targeting rAd to alternative DC surface molecules, DC infected with all currently tested rAd constitute potent immunostimulators. Our study provides new insights into the interactions between two highly promising vaccine components, rAd and DC, and indicates that their combination into one vaccine may be very advantageous for the stimulation of T-cell immunity.     

Induction of antitumor immunity by vaccination of dendritic cells transfected with MUC1 RNA

Dendritic cells (DC) are potent APCs. In this study, murine bone marrow-derived DC were transfected with RNA encoding the MUC1 Ag that is aberrantly overexpressed in human breast and other carcinomas. The MUC1 RNA-transfected DC exhibited cell surface expression of MUC1 and costimulatory molecules. After injection at the base of the tail, the transfected DC were detectable in inguinal lymph nodes by dual immunochemical staining. Vaccination of wild-type mice with MUC1 RNA-transfected DC induced anti-MUC1 immune responses against MUC1-positive MC38/MUC1, but not MUC1-negative, tumor cells. Mice immunized with the transfected DC were protected against challenge with MC38/MUC1 tumor cells. Furthermore, mice with established MC38/MUC1 tumors were eliminated after receiving the vaccination. CTLs isolated from mice immunized with the transfected DC exhibited specific cytolytic activity against MC38/MUC1 tumor cells. In contrast to these findings, there was little if any anti-MUC1 immunity induced with the transfected DC in MUC1 transgenic (MUC1.Tg) mice. However, coadministration of the transfected DC and IL-12 reversed the unresponsiveness to MUC1 Ag in MUC1.Tg mice and induced MUC1-specific immune responses. These findings demonstrate that vaccination of DC transfected with MUC1 RNA and IL-12 reverses tolerance to MUC1 and induces immunity against MUC1-positive tumors.     

Short-term culture of myeloid leukemic cells allows efficient transduction by adenoviral vectors

BACKGROUND: Ex vivo gene therapy of acute myeloid leukemia (AML) requires efficient transduction of leukemic cells. Recombinant adenovirus has been reported to be a poorly efficient vector in leukemic cells. We investigated leukemic cell culture as a possible method of improving the efficacy of this vector. METHODS: Leukemic cell lines and primary cultured AML cells were incubated with adenoviral vectors carrying GFP, LacZ, or IL-12 cDNA. Transduction efficiency was evaluated by measuring adenoviral genome copy number and transgene expression in leukemic cells. The expression of the coxsackie/adenovirus receptor (CAR), CD29, CD49e, and CD51/61 was measured, as was the effect of blocking integrin on adenoviral transduction. RESULTS: Increasing the multiplicity of infection (MOI) to 300 plaque-forming units per cell enhanced transduction of leukemic cell lines and to a lesser degree of AML cells. Analysis of adenoviral genome copy per cell showed only a partial correlation between gene transfer efficiency and transgene expression. Culture of AML cells for 3 days prior to adenoviral transduction increased both adenoviral copy number per cell and the percentage of transgene-expressing cells. CD29, CD49e, and CD51/61 but not CAR expression increased in cultured AML cells between days 0 and 3 and integrin-blocking experiments showed inhibition of transduction in two of four AML samples tested. CONCLUSIONS: Efficient ex vivo gene transfer in primary cultured AML cells can be achieved by short-term culture of leukemic cells prior to gene transfer with adenoviral vectors at a high MOI. This effect appears to be at least partially mediated by enhanced integrin expression.     

Prolonged maturation and enhanced transduction of dendritic cells migrated from human skin explants after in situ delivery of CD40-targeted adenoviral vectors

Therapeutic tumor vaccination with viral vectors or naked DNA, carrying the genetic code for tumor-associated Ags, critically depends on the in vivo transduction of dendritic cells (DC). Transfection of predominantly nonprofessional APC and only small numbers of DC may hamper proper T cell activation. Aim of this study was, therefore, the targeted, selective, and enhanced in situ transduction of DC. A human skin explant model was used to explore targeted transduction of cutaneous DC after intradermal injection of a bispecific Ab conjugate to link adenoviral (Ad) vectors directly to CD40 on the DC surface. A significantly enhanced transduction efficiency and selectivity, and an increased activation state of migrating DC were thus achieved. Moreover, DC transduced by CD40-targeted Ad maintained their Ag-specific CTL-stimulatory ability for up to 1 wk after the start of migration, in contrast to DC transduced by untargeted Ad, which had lost this capacity by that time. Because DC targeting in vivo might obviate the need for the in vitro culture of autologous DC for adoptive transfer, CD40-targeted Ad vectors constitute a promising new vaccine modality for tumor immunotherapy.     

Endogenous granulocyte-macrophage colony-stimulating factor overexpression in vivo results in the long-term recruitment of a distinct dendritic cell population with enhanced immunostimulatory function

GM-CSF is critical for dendritic cell (DC) survival and differentiation in vitro. To study its effect on DC development and function in vivo, we used a gene transfer vector to transiently overexpress GM-CSF in mice. We found that up to 24% of splenocytes became CD11c+ and the number of DC increased up to 260-fold to 3 x 10(8) cells. DC numbers remained substantially elevated even 75 days after treatment. The DC population was either CD8alpha+CD4- or CD8alpha-CD4- but not CD8alpha+CD4+ or CD8alpha-CD4+. This differs substantially from subsets recruited in normal or Flt3 ligand-treated mice or using GM-CSF protein injections. GM-CSF-recruited DC secreted extremely high levels of TNF-alpha compared with minimal amounts in DC from normal or Flt3 ligand-treated mice. Recruited DC also produced elevated levels of IL-6 but almost no IFN-gamma. GM-CSF DC had robust immune function compared with controls. They had an increased rate of Ag capture and caused greater allogeneic and Ag-specific T cell stimulation. Furthermore, GM-CSF-recruited DC increased NK cell lytic activity after coculture. The enhanced T cell and NK cell immunostimulation by GM-CSF DC was in part dependent on their secretion of TNF-alpha. Our findings show that GM-CSF can have an important role in DC development and recruitment in vivo and has potential application to immunotherapy in recruiting massive numbers of DC with enhanced ability to activate effector cells.     

Cancer Associated Aberrant Protein O-Glycosylation Can Modify Antigen Processing and Immune Response

Aberrant glycosylation of mucins and other extracellular proteins is an important event in carcinogenesis and the resulting cancer associated glycans have been suggested as targets in cancer immunotherapy. We assessed the role of O-linked GalNAc glycosylation on antigen uptake, processing, and presentation on MHC class I and II molecules. The effect of GalNAc O-glycosylation was monitored with a model system based on ovalbumin (OVA)-MUC1 fusion peptides (+/− glycosylation) loaded onto dendritic cells co-cultured with IL-2 secreting OVA peptide-specific T cell hybridomas. To evaluate the in vivo response to a cancer related tumor antigen, Balb/c or B6.Cg(CB)-Tg(HLA-A/H2-D)2Enge/J (HLA-A2 transgenic) mice were immunized with a non-glycosylated or GalNAc-glycosylated MUC1 derived peptide followed by comparison of T cell proliferation, IFN-γ release, and antibody induction. GalNAc-glycosylation promoted presentation of OVA-MUC1 fusion peptides by MHC class II molecules and the MUC1 antigen elicited specific Ab production and T cell proliferation in both Balb/c and HLA-A2 transgenic mice. In contrast, GalNAc-glycosylation inhibited the presentation of OVA-MUC1 fusion peptides by MHC class I and abolished MUC1 specific CD8+ T cell responses in HLA-A2 transgenic mice. GalNAc glycosylation of MUC1 antigen therefore facilitates uptake, MHC class II presentation, and antibody response but might block the antigen presentation to CD8+ T cells.     

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.     

Three different vaccines based on the 140-amino acid MUC1 peptide with seven tandemly repeated tumor-specific epitopes elicit distinct immune effector mechanisms in wild-type versus MUC1-transgenic mice with different potential for tumor rejection

Low-frequency CTL and low-titer IgM responses against tumor-associated Ag MUC1 are present in cancer patients but do not prevent cancer growth. Boosting MUC1-specific immunity with vaccines, especially effector mechanisms responsible for tumor rejection, is an important goal. We studied immunogenicity, tumor rejection potential, and safety of three vaccines: 1) MUC1 peptide admixed with murine GM-CSF as an adjuvant; 2) MUC1 peptide admixed with adjuvant SB-AS2; and 3) MUC1 peptide-pulsed dendritic cells (DC). We examined the qualitative and quantitative differences in humoral and T cell-mediated MUC1-specific immunity elicited in human MUC1-transgenic (Tg) mice compared with wild-type (WT) mice. Adjuvant-based vaccines induced MUC1-specific Abs but failed to stimulate MUC1-specific T cells. MUC1 peptide with GM-CSF induced IgG1 and IgG2b in WT mice but only IgM in MUC1-Tg mice. MUC1 peptide with SB-AS2 induced high-titer IgG1, IgG2b, and IgG3 Abs in both WT and MUC1-Tg mice. Induction of IgG responses was T cell independent and did not have any effect on tumor growth. MUC1 peptide-loaded DC induced only T cell immunity. If injected together with soluble peptide, the DC vaccine also triggered Ab production. Importantly, the DC vaccine elicited tumor rejection responses in both WT and MUC1-Tg mice. These responses correlated with the induction of MUC1-specific CD4+ and CD8+ T cells in WT mice, but only CD8(+) T cells in MUC1-Tg mice. Even though MUC1-specific CD4+ T cell tolerance was not broken, the capacity of MUC1-Tg mice to reject tumor was not compromised.     

HIV-1 capture and antigen presentation by dendritic cells: enhanced viral capture does not correlate with better T cell activation

During HIV-1 infection, dendritic cells (DC) facilitate dissemination of HIV-1 while trying to trigger adaptive antiviral immune responses. We examined whether increased HIV-1 capture in DC matured with LPS results in more efficient Ag presentation to HIV-1-specific CD4(+) and CD8(+) T cells. To block the DC-mediated trans-infection of HIV-1 and maximize Ag loading, we also evaluated a noninfectious integrase-deficient HIV-1 isolate, HIV(NL4-3ΔIN). We showed that higher viral capture of DC did not guarantee better Ag presentation or T cell activation. Greater HIV(NL4-3) uptake by fully LPS-matured DC resulted in higher viral transmission to target cells but poorer stimulation of HIV-1-specific CD4(+) and CD8(+) T cells. Conversely, maturation of DC with LPS during, but not before, viral loading enhanced both HLA-I and HLA-II HIV-1-derived Ag presentation. In contrast, DC maturation with the clinical-grade mixture consisting of IL-1β, TNF-α, IL-6, and PGE(2) during viral uptake only stimulated HIV-1-specific CD8(+) T cells. Hence, DC maturation state, activation stimulus, and time lag between DC maturation and Ag loading impact HIV-1 capture and virus Ag presentation. Our results demonstrate a dissociation between the capacity to capture HIV-1 and to present viral Ags. Integrase-deficient HIV(NL4-3ΔIN) was also efficiently captured and presented by DC through the HLA-I and HLA-II pathways but in the absence of viral dissemination. HIV(NL4-3ΔIN) seems to be an attractive candidate to be explored. These results provide new insights into DC biology and have implications in the optimization of DC-based immunotherapy against HIV-1 infection.     

Murine Flt3 ligand expands distinct dendritic cells with both tolerogenic and immunogenic properties

Human Flt3 ligand can expand dendritic cells (DC) and enhance immunogenicity in mice. However, little is known about the effects of murine Flt3 ligand (mFlt3L) on mouse DC development and function. We constructed a vector to transiently overexpress mFlt3L in mice. After a single treatment, up to 44% of splenocytes became CD11c(+) and the total number of DC increased 100-fold. DC expansion effects lasted for >35 days. mFlt3L DC were both phenotypically and functionally distinct. They had increased expression of MHC and costimulatory molecules and expressed elevated levels of B220 and DEC205 but had minimal CD4 staining. mFlt3L DC also had a markedly altered cytokine profile, including lowered secretion of IL-6, IL-10, IFN-gamma, and TNF-alpha, but had a slightly increased capacity to stimulate T cells in vitro. However, in a variety of in vivo models, DC expanded by mFlt3L induced tolerogenic effects on T cells. Adoptive transfer of Ag-pulsed mFlt3L splenic DC to naive mice actually caused faster rates of tumor growth and induced minimal CTL compared with control DC. mFlt3L also failed to protect against tumors in which human Flt3 ligand was protective, but depletion of CD4(+) T cells restored tumor protection. Our findings 1) demonstrate that mFlt3L has distinct effects on DC development, 2) suggest an important role for mFlt3L in generating DC that have tolerogenic effects on T cells, and 3) may have application in immunotherapy in generating massive numbers of DC for an extended duration.