The relative efficiency of acquisition of MHC:peptide complexes and cross-presentation depends on dendritic cell type

Intercellular exchange of MHC molecules has been reported between many cells, including professional and nonprofessional APCs. This phenomenon may contribute to T cell immunity to pathogens. In this study, we addressed whether the transfer of MHC class I:peptide complexes between cells plays a role in T cell responses and compare this to conventional cross-presentation. We observed that dsRNA-matured bone marrow-derived dendritic cells (BMDCs) acquired peptide:MHC complexes from other BMDCs either pulsed with OVA(257-264) peptide, soluble OVA, or infected with a recombinant adenovirus expressing OVA. In addition, BMDCs were capable of acquiring MHC:peptide complexes from epithelial cells. Spleen-derived CD8alpha(+) and CD8alpha(-) dendritic cells (DCs) also acquired MHC:peptide complexes from BMDCs pulsed with OVA(257-264) peptide. However, the efficiency of acquisition by these ex vivo derived DCs is much lower than acquisition by BMDC. In all cases, the acquired MHC:peptide complexes were functional in that they induced Ag-specific CD8(+) T cell proliferation. The efficiency of MHC transfer was compared with cross-presentation for splenic CD8alpha(+) and CD8alpha(-) as well as BMDCs. CD8alpha(+) DCs were more efficient at inducing T cell proliferation when they acquired Ag via cross-presentation, the opposite was observed for BMDCs and splenic CD8alpha(-) DCs. We conclude from these observations that the relative efficiency of MHC transfer vs cross-presentation differs markedly between different DC subsets.     

Phosphatidylserine receptor-mediated recognition of archaeosome adjuvant promotes endocytosis and MHC class I cross-presentation of the entrapped antigen by phagosome-to-cytosol transport and classical processing

Archaeal isopranoid glycerolipid vesicles (archaeosomes) serve as strong adjuvants for cell-mediated responses to entrapped Ag. We analyzed the processing pathway of OVA entrapped in archaeosomes composed of Methanobrevibacter smithii lipids, high in archaetidylserine (OVA-archaeosomes). In vitro, OVA-archaeosomes stimulated spleen cells from OVA-TCR-transgenic mice, D011.10 (CD4(+) cells expressing OVA(323-339) TCR) or OT1 (>90% CD8(+) OVA(257-264) cells), indicating both MHC class I and II presentations. In vivo, when naive (Thy1.2(+)) CFSE-labeled OT1 cells were transferred into OVA-archaeosome-immunized Thy 1.1(+) recipient mice, there was profound accumulation and cycling of donor-specific cells, and differentiation of H-2K(b)Ova(257-264) CD8(+) T cells into CD44(high)CD62L(low) effectors. Both macrophages and dendritic cells (DCs) efficiently cross-presented OVA-archaeosomes on MHC class I. Blocking phagocytosis by phosphatidylserine-specific receptor agonists strongly inhibited MHC class I presentation of OVA-archaeosomes, whereas blocking mannose receptors or FcRs lacked effect, indicating specific recognition of the archaetidylserine head group of M. smithii lipids by APCs. In addition, inhibitors of endosomal acidification blocked MHC class I processing of OVA-archaeosomes, whereas endosomal protease inhibitors lacked effect, suggesting acidification-dependent phagosome-to-cytosol diversion. Proteasomal inhibitors blocked OVA-archaeosome MHC class I presentation, confirming cytosolic processing. Both in vitro and in vivo, OVA-archaeosome MHC class I presentation required TAP. Ag-free archaeosomes also activated DC costimulation and cytokine production, without overt inflammation. Phosphatidylserine-specific receptor-mediated endocytosis is a mechanism of apoptotic cell clearance and DCs cross-present Ags sampled from apoptotic cells. Our results reveal the novel ability of archaeosomes to exploit this mechanism for cytosolic MHC class I Ag processing, and provide an effective particulate vaccination strategy.     

A marked reduction in priming of cytotoxic CD8+ T cells mediated by stress-induced glucocorticoids involves multiple deficiencies in cross-presentation by dendritic cells

Protracted psychological stress elevates circulating glucocorticoids, which can suppress CD8(+) T cell-mediated immunity, but the mechanisms are incompletely understood. Dendritic cells (DCs), required for initiating CTL responses, are vulnerable to stress/corticosterone, which can contribute to diminished CTL responses. Cross-priming of CD8(+) T cells by DCs is required for initiating CTL responses against many intracellular pathogens that do not infect DCs. We examined the effects of stress/corticosterone on MHC class I (MHC I) cross-presentation and priming and show that stress/corticosterone-exposed DCs have a reduced ability to cross-present OVA and activate MHC I-OVA(257-264)-specific T cells. Using a murine model of psychological stress and OVA-loaded β(2)-microglobulin knockout "donor" cells that cannot present Ag, DCs from stressed mice induced markedly less Ag-specific CTL proliferation in a glucocorticoid receptor-dependent manner, and endogenous in vivo T cell cytolytic activity generated by cross-presented Ag was greatly diminished. These deficits in cross-presentation/priming were not due to altered Ag donation, Ag uptake (phagocytosis, receptor-mediated endocytosis, or fluid-phase uptake), or costimulatory molecule expression by DCs. However, proteasome activity in corticosterone-treated DCs or splenic DCs from stressed mice was partially suppressed, which limits formation of antigenic peptide-MHC I complexes. In addition, the lymphoid tissue-resident CD11b(-)CD24(+)CD8α(+) DC subset, which carries out cross-presentation/priming, was preferentially depleted in stressed mice. At the same time, CD11b(-)CD24(+)CD8α(-) DC precursors were increased, suggesting a block in development of CD8α(+) DCs. Therefore, glucocorticoid-induced changes in both the cellular composition of the immune system and intracellular protein degradation contribute to impaired CTL priming in stressed mice.     

Acquisition of MHC:Peptide Complexes by Dendritic Cells Contributes to the Generation of Antiviral CD8+ T Cell Immunity In Vivo

There is an increasing body of evidence suggesting that the transfer of preformed MHC class I:peptide complexes between a virus-infected cell and an uninfected APC, termed cross-dressing, represents an important mechanism of Ag presentation to CD8(+) T cells in host defense. However, although it has been shown that memory CD8(+) T cells can be activated by uninfected dendritic cells (DCs) cross-dressed by Ag from virus-infected parenchymal cells, it is unknown whether conditions exist during virus infection in which naive CD8(+) T cells are primed and differentiate to cytolytic effectors through cross-dressing, and indeed which DC subset would be responsible. In this study, we determine whether the transfer of MHC class I:peptide complexes between infected and uninfected murine DC plays a role in CD8(+) T cell priming to viral Ags in vivo. We show that MHC class I:peptide complexes from peptide-pulsed or virus-infected DCs are indeed acquired by splenic CD8α(-) DCs in vivo. Furthermore, the acquired MHC class I:peptide complexes are functional in that they induced Ag-specific CD8(+) T cell effectors with cytolytic function. As CD8α(-) DCs are poor cross-presenters, this may represent the main mechanism by which CD8α(-) DCs present exogenously encountered Ag to CD8(+) T cells. The sharing of Ag as preformed MHC class I:peptide complexes between infected and uninfected DCs without the restraints of Ag processing may have evolved to accurately amplify the response and also engage multiple DC subsets critical in the generation of strong antiviral immunity.     

CpG-DNA aided cross-priming by cross-presenting B cells

Covalent linkage of immunostimulatory CpG-DNA to OVA (CpG-OVA complex) results in CpG-DNA-aided cross-presentation of OVA by dendritic cells (DCs). In this study, we analyzed the thesis that CpG-OVA complexes may be cross-presented by B cells to route internalized Ag into the class I MHC presentation pathway. First, we describe that conjugation of CpG-DNA to OVA enhances up to 40-fold internalization of OVA by B cells, which in turn generate the CD8 T cell epitope SIINFEKL complexed to MHC class I, albeit less efficiently than DCs. Furthermore, upon internalization, CpG-DNA conjugated to OVA stimulates B cells to up-regulate costimulatory molecules and cytokines including IL-12. Adoptive transfer of CpG-OVA complex-loaded wild-type B cells cross-primes naive CD8 T cells both in wild-type mice and in MyD88-deficient mice. Overall, these findings disclose attributes of B cells, including cross-presentation of exogenous Ag and cross-priming of naive CD8 T cells that hitherto have been considered as hallmarks restricted to DCs.     

Protection against influenza A virus by memory CD8 T cells requires reactivation by bone marrow-derived dendritic cells

Influenza A virus is the causative agent of an acute inflammatory disease of the airway. Although Abs can prevent infection, disease and death can be prevented by T cell-mediated immunity. Recently, we showed that protection against lethal influenza A (PR8/34) virus infection is mediated by central memory CD8 T cells (T(CM)). In this study, using relB(-/-) mice we began to investigate the role of bone marrow (BM)-derived dendritic cells (DCs) in the mechanism of protection. We found that in the absence of functional DCs, memory CD8 T cells specific for the nucleoprotein epitope (NP(366-374)) fail to protect even after adoptive transfer into naive recipients. Through an analysis of Ag uptake, activation of memory CD8 T cells, and display of peptide/MHC complex by DCs in draining LNs and spleen early after virus infection, we established that lack of protection is associated with defective Ag presentation by BM-derived DCs and defective homing of memory T cells in the lymph nodes draining the airway tract. Collectively, the data suggest that protection against the influenza A virus requires that memory CD8 T cells be reactivated by Ag presented by BM-derived DCs in the lymph nodes draining the site of infection. They also imply that protection depends both on the characteristics of systemic adaptive immunity and on the coordinated interplay between systemic and local immunity.     

Cutting edge: langerin/CD207 receptor on dendritic cells mediates efficient antigen presentation on MHC I and II products in vivo

The targeted delivery of Ags to dendritic cell (DCs) in vivo greatly improves the efficiency of Ag presentation to T cells and allows an analysis of receptor function. To evaluate the function of Langerin/CD207, a receptor expressed by subsets of DCs that frequently coexpress the DEC205/CD205 receptor, we genetically introduced OVA into the C terminus of anti-receptor Ab H chains. Taking advantage of the new L31 mAb to the extracellular domain of mouse Langerin, we find that the hybrid Ab targets appropriate DC subsets in draining lymph nodes and spleen. OVA is then presented efficiently to CD8(+) and CD4(+) T cells in vivo, which undergo 4-8 cycles of division in 3 days. Peptide MHC I and II complexes persist for days. Dose response studies indicate only modest differences between Langerin and DEC receptors in these functions. Thus, Langerin effectively mediates Ag presentation.     

The adjuvanticity of a mannosylated antigen reveals TLR4 functionality essential for subset specialization and functional maturation of mouse dendritic cells

The evidence that dendritic cell (DC) subsets produce differential cytokines in response to specific TLR stimulation is robust. However, the role of TLR stimulation in Ag presentation and phenotypic maturation among DC subsets is not clear. Through the adjuvanticity of a novel mannosylated Ag, mannosylated dendrimer OVA (MDO), as a pathogen-associated molecular pattern Ag, we characterized the functionality of GM-CSF/IL-4-cultured bone marrow DC and Flt3 ligand (Flt3-L) DC subsets by Ag presentation and maturation assays. It was demonstrated that both bone marrow DCs and Flt3-L DCs bound, processed, and presented MDO effectively. However, while Flt3-L CD24(high) (conventional CD8(+) equivalent) and CD11b(high) (CD8(-) equivalent) DCs were adept at MDO processing by MHC class I and II pathways, respectively, CD45RA(+) plasmacytoid DCs presented MDO poorly to T cells. Successful MDO presentation was largely dependent on competent TLR4 for Ag localization and morphological/phenotypic maturation of DC subsets, despite the indirect interaction of MDO with TLR4. Furthermore, Toll/IL-1 receptor-domain-containing adaptor-inducing IFN-beta, but not MyD88, as a TLR4 signaling modulator was indispensable for MDO-induced DC maturation and Ag presentation. Taken together, our findings suggest that DC subsets differentially respond to a pathogen-associated molecular pattern-associated Ag depending on the intrinsic programming and TLRs expressed. Optimal functionality of DC subsets in Ag presentation necessitates concomitant TLR signaling critical for efficient Ag localization and processing.     

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.     

A role for a melanosome transport signal in accessing the MHC class II presentation pathway and in eliciting CD4+ T cell responses

Melanosomal membrane proteins are frequently recognized by the immune system of patients with melanoma and vitiligo. Melanosomal glycoproteins are transported to melanosomes by a dileucine-based melanosomal transport signal (MTS). To investigate whether this sorting signal could be involved in presentation of melanosome membrane proteins to the immune system, we devised a fusion construct containing the MTS from the mouse brown locus product gp75/tyrosinase-related protein-1 and full-length OVA as a reporter Ag. The fusion protein was expressed as an intracellular membrane protein, sorted to the endocytic pathway, processed, and presented by class II MHC molecules. DNA immunization with this construct elicited CD4+ T cell proliferative responses in vivo. Ag presentation and T cell responses in vitro and in vivo required a functional MTS. Mutations of either the upstream leucine in MTS or elimination of the entire MTS negated in vitro Ag presentation and in vivo T cell responses. In a mouse melanoma model, DNA immunization with MTS constructs protected mice from tumor challenge in a CD4+ T cell-dependent manner, but complete deletion of MTS decreased tumor rejection. Therefore, MTS can target epitopes to the endocytic pathway leading to presentation by class II MHC molecules to helper T cells.     

CD13 regulates dendritic cell cross-presentation and T cell responses by inhibiting receptor-mediated antigen uptake

Dendritic cell (DC) Ag cross-presentation is generally associated with immune responses to tumors and viral Ags, and enhancement of this process is a focus of tumor vaccine design. In this study, we found that the myeloid cell surface peptidase CD13 is highly and specifically expressed on the subset of DCs responsible for cross-presentation, the CD8(+) murine splenic DCs. In vivo studies indicated that lack of CD13 significantly enhanced T cell responses to soluble OVA Ag, although development, maturation, and Ag processing and presentation of DCs are normal in CD13KO mice. In vitro studies showed that CD13 regulates receptor-mediated, dynamin-dependent endocytosis of Ags such as OVA and transferrin but not fluid-phase or phagocytic Ag uptake. CD13 and Ag are cointernalized in DCs, but CD13 did not coimmunoprecipitate with Ag receptors, suggesting that CD13 does not control internalization of specific receptors but regulates endocytosis at a more universal level. Mechanistically, we found that phosphorylation of the endocytic regulators p38MAPK and Akt was dysregulated in CD13KO DCs, and blocking of these kinases perturbed CD13-dependent endocytic uptake. Therefore, CD13 is a novel endocytic regulator that may be exploited to enhance Ag uptake and T cell activation to improve the efficacy of tumor-targeted vaccines.     

Calcineurin inhibitors block MHC-restricted antigen presentation in vivo

APCs, like T cells, are affected by calcineurin inhibitors. In this study, we show that calcineurin inhibitors efficiently block MHC-restricted exogenous Ag presentation in vivo. Mice were injected with clinical doses of tacrolimus (FK-506) followed by soluble OVA, and dendritic cells (DCs) were isolated from lymph nodes and spleens. The efficacy of OVA peptide presentation by DCs was evaluated using OVA-specific CD8 and CD4 T cells. Tacrolimus inhibited both class I- and class II-restricted DC presentation of OVA to T cells. Tacrolimus also inhibited both class I- and class II-restricted presentation of OVA in peritoneal macrophages isolated from mice injected with tacrolimus followed by soluble OVA. Tacrolimus-treated peritoneal macrophages, however, were able to present synthetic OVA peptide, SIINFEKL. Inclusion of cyclosporine A to biodegradable microspheres containing OVA greatly reduced their capacity to induce OVA-specific CTL response in mice. These findings provide novel insight into the mode of action of calcineurin inhibitors and have important implications for clinical immunosuppression regimens.     

Immune complex-loaded dendritic cells are superior to soluble immune complexes as antitumor vaccine

Dendritic cells (DCs) play an important role in the induction of T cell responses. Fc gammaRs, expressed on DCs, facilitate the uptake of complexed Ag, resulting in efficient MHC class I and MHC class II Ag presentation and DC maturation. In the present study, we show that prophylactic immunization with DCs loaded with Ag-IgG immune complexes (ICs) leads to efficient induction of tumor protection in mice. Therapeutic vaccinations strongly delay tumor growth or even prevent tumors from growing out. By depleting CD4+ and CD8+ cell populations before tumor challenge, we identify CD8+ cells as the main effector cells involved in tumor eradication. Importantly, we show that DCs that are preloaded in vitro with ICs are at least 1000-fold more potent than ICs injected directly into mice or DCs loaded with the same amount of noncomplexed protein. The contribution of individual Fc gammaRs to Ag presentation, T cell response induction, and induction of tumor protection was assessed. We show that Fc gammaRI and Fc gammaRIII are capable of enhancing MHC class I-restricted Ag presentation to CD8+ T cells in vitro and that these activating Fc gammaRs on DCs are required for efficient priming of Ag-specific CD8+ cells in vivo and induction of tumor protection. These findings show that targeting ICs via the activating Fc gammaRs to DCs in vitro is superior to direct IC vaccination to induce protective tumor immunity in vivo.     

CD1 molecules efficiently present antigen in immature dendritic cells and traffic independently of MHC class II during dendritic cell maturation

Upon exposure to Ag and inflammatory stimuli, dendritic cells (DCs) undergo a series of dynamic cellular events, referred to as DC maturation, that involve facilitated peptide Ag loading onto MHC class II molecules and their subsequent transport to the cell surface. Besides MHC molecules, human DCs prominently express molecules of the CD1 family (CD1a, -b, -c, and -d) and mediate CD1-dependent presentation of lipid and glycolipid Ags to T cells, but the impact of DC maturation upon CD1 trafficking and Ag presentation is unknown. Using monocyte-derived immature DCs and those stimulated with TNF-alpha for maturation, we observed that none of the CD1 isoforms underwent changes in intracellular trafficking that mimicked MHC class II molecules during DC maturation. In contrast to the striking increase in surface expression of MHC class II on mature DCs, the surface expression of CD1 molecules was either increased only slightly (for CD1b and CD1c) or decreased (for CD1a). In addition, unlike MHC class II, DC maturation-associated transport from lysosomes to the plasma membrane was not readily detected for CD1b despite the fact that both molecules were prominently expressed in the same MIIC lysosomal compartments before maturation. Consistent with this, DCs efficiently presented CD1b-restricted lipid Ags to specific T cells similarly in immature and mature DCs. Thus, DC maturation-independent pathways for lipid Ag presentation by CD1 may play a crucial role in host defense, even before DCs are able to induce maximum activation of peptide Ag-specific T cells.     

Archaeosomes induce long-term CD8+ cytotoxic T cell response to entrapped soluble protein by the exogenous cytosolic pathway, in the absence of CD4+ T cell help

The unique ether glycerolipids of ARCHAEA: can be formulated into vesicles (archaeosomes) with strong adjuvant activity for MHC class II presentation. Herein, we assess the ability of archaeosomes to facilitate MHC class I presentation of entrapped protein Ag. Immunization of mice with OVA entrapped in archaeosomes resulted in a potent Ag-specific CD8(+) T cell response, as measured by IFN-gamma production and cytolytic activity toward the immunodominant CTL epitope OVA(257-264). In contrast, administration of OVA with aluminum hydroxide or entrapped in conventional ester-phospholipid liposomes failed to evoke significant CTL response. The archaeosome-mediated CD8(+) T cell response was primarily perforin dependent because CTL activity was undetectable in perforin-deficient mice. Interestingly, a long-term CTL response was generated with a low Ag dose even in CD4(+) T cell deficient mice, indicating that the archaeosomes could mediate a potent T helper cell-independent CD8(+) T cell response. Macrophages incubated in vitro with OVA archaeosomes strongly stimulated cytokine production by OVA-specific CD8(+) T cells, indicating that archaeosomes efficiently delivered entrapped protein for MHC class I presentation. This processing of Ag was Brefeldin A sensitive, suggesting that the peptides were transported through the endoplasmic reticulum and presented by the cytosolic MHC class I pathway. Finally, archaeosomes induced a potent memory CTL response to OVA even 154 days after immunization. This correlated to strong Ag-specific up-regulation of CD44 on splenic CD8(+) T cells. Thus, delivery of proteins in self-adjuvanting archaeosomes represents a novel strategy for targeting exogenous Ags to the MHC class I pathway for induction of CTL response.     

Acidosis improves uptake of antigens and MHC class I-restricted presentation by dendritic cells

It is widely appreciated that inflammatory responses in peripheral tissues are usually associated to the development of acidic microenvironments. Despite this, there are few studies aimed to analyze the effect of extracellular pH on immune cell functions. We analyzed the impact of acidosis on the behavior of dendritic cells (DCs) derived from murine bone marrow. We found that extracellular acidosis (pH 6.5) markedly stimulated the uptake of FITC-OVA, FITC-dextran, and HRP by DCs. In fact, to reach similar levels of endocytosis, DCs cultured at pH 7.3 required concentrations of Ag in the extracellular medium almost 10-fold higher compared with DCs cultured at pH 6.5. Not only the endocytic capacity of DCs was up-regulated by extracellular acidosis, but also the expression of CD11c, MHC class II, CD40, and CD86 as well as the acquisition of extracellular Ags by DCs for MHC class I-restricted presentation. Importantly, DCs pulsed with Ag under acidosis showed an improved efficacy to induce both specific CD8(+) CTLs and specific Ab responses in vivo. Our results suggest that extracellular acidosis improves the Ag-presenting capacity of DCs.     

The neonatal FcR-mediated presentation of immune-complexed antigen is associated with endosomal and phagosomal pH and antigen stability in macrophages and dendritic cells

The FcγRs found on macrophages (Ms) and dendritic cells (DCs) efficiently facilitate the presentation or cross-presentation of immune-complexed Ags to T cells. We found that the MHC class I-related neonatal FcR for IgG (FcRn) in both Ms and DCs failed to have a strong effect on the cross-presentation of immune complex (IC) OVA Ag to CD8(+) T cells. Interestingly, endosomal FcRn enhanced the presentation of the monomeric OVA-IC to CD4(+) T cells robustly, whereas FcRn in phagosomes exerted distinctive effects on Ag presentation between Ms and DCs. The presentation of phagocytosed OVA-ICs to CD4(+) T cells was considerably enhanced on wild-type versus FcRn-deficient Ms, but was not affected in FcRn-deficient DCs. This functional discrepancy was associated with the dependence of IgG-FcRn binding in an acidic pH. Following phagocytosis, the phagosomal pH dropped rapidly to <6.5 in Ms but remained in the neutral range in DCs. This disparity in pH determined the rate of degradation of phagocytosed ICs. Thus, our findings reveal that FcRn expression has a different effect on Ag processing and presentation of ICs to CD4(+) T cells in the endosomal versus phagosomal compartments of Ms versus DCs.     

ISCOMATRIX adjuvant combines immune activation with antigen delivery to dendritic cells in vivo leading to effective cross-priming of CD8+ T cells

Cancer vaccines aim to induce CTL responses against tumors. Challenges for vaccine design are targeting Ag to dendritic cells (DCs) in vivo, facilitating cross-presentation, and conditioning the microenvironment for Th1 type immune responses. In this study, we report that ISCOM vaccines, which consist of ISCOMATRIX adjuvant and protein Ag, meet these challenges. Subcutaneous injection of an ISCOM vaccine in mice led to a substantial influx and activation of innate and adaptive immune effector cells in vaccine site-draining lymph nodes (VDLNs) as well as IFN-γ production by NK and NKT cells. Moreover, an ISCOM vaccine containing the model Ag OVA (OVA/ISCOM vaccine) was efficiently taken up by CD8α(+) DCs in VDLNs and induced their maturation and IL-12 production. Adoptive transfer of transgenic OT-I T cells revealed highly efficient cross-presentation of the OVA/ISCOM vaccine in vivo, whereas cross-presentation of soluble OVA was poor even at a 100-fold higher concentration. Cross-presenting activity was restricted to CD8α(+) DCs in VDLNs, whereas Langerin(+) DCs and CD8α(-) DCs were dispensable. Remarkably, compared with other adjuvant systems, the OVA/ISCOM vaccine induced a high frequency of OVA-specific CTLs capable of tumor cell killing in different tumor models. Thus, ISCOM vaccines combine potent immune activation with Ag delivery to CD8α(+) DCs in vivo for efficient induction of CTL responses.     

GM-CSF is an essential regulator of T cell activation competence in uterine dendritic cells during early pregnancy in mice

Uterine dendritic cells (DCs) are critical for activating the T cell response mediating maternal immune tolerance of the semiallogeneic fetus. GM-CSF (CSF2), a known regulator of DCs, is synthesized by uterine epithelial cells during induction of tolerance in early pregnancy. To investigate the role of GM-CSF in regulating uterine DCs and macrophages, Csf2-null mutant and wild-type mice were evaluated at estrus, and in the periconceptual and peri-implantation periods. Immunohistochemistry showed no effect of GM-CSF deficiency on numbers of uterine CD11c(+) cells and F4/80(+) macrophages at estrus or on days 0.5 and 3.5 postcoitum, but MHC class II(+) and class A scavenger receptor(+) cells were fewer. Flow cytometry revealed reduced CD80 and CD86 expression by uterine CD11c(+) cells and reduced MHC class II in both CD11c(+) and F4/80(+) cells from GM-CSF-deficient mice. CD80 and CD86 were induced in Csf2(-/-) uterine CD11c(+) cells by culture with GM-CSF. Substantially reduced ability to activate both CD4(+) and CD8(+) T cells in vivo was evident after delivery of OVA Ag by mating with Act-mOVA males or transcervical administration of OVA peptides. This study shows that GM-CSF regulates the efficiency with which uterine DCs and macrophages activate T cells, and it is essential for optimal MHC class II- and class I-mediated indirect presentation of reproductive Ags. Insufficient GM-CSF may impair generation of T cell-mediated immune tolerance at the outset of pregnancy and may contribute to the altered DC profile and dysregulated T cell tolerance evident in infertility, miscarriage, and preeclampsia.