Both dendritic cells and macrophages can stimulate naive CD8 T cells in vivo to proliferate, develop effector function, and differentiate into memory cells

The generation of T cell immunity requires the acquisition and presentation of Ag on bone marrow-derived APCs. Dendritic cells (DC) are believed to be the most potent bone marrow-derived APCs, and the only ones that can stimulate naive T cells to productively respond to Ags. Because macrophages (Mphi) are bone marrow-derived APCs that are also found in tissues and lymphoid organs, can acquire and present Ag, and can express costimulatory molecules, we have investigated their potential to stimulate primary T cell responses in vivo. We find that both injected Mphi and DCs can migrate from peripheral tissues or blood into lymphoid organs. Moreover, injection of peptide-pulsed Mphi or DCs into mice stimulates CD8 T cells to proliferate, express effector functions including cytokine production and cytolysis, and differentiate into long-lived memory cells. Mphi and DCs stimulate T cells directly without requiring cross-presentation of Ag on host APCs. Therefore, more than one type of bone marrow-derived APC has the potential to prime T cell immunity. In contrast, another bone marrow-derived cell, the T lymphocyte, although capable of presenting Ag and homing to the T cell areas of lymphoid organs, is unable to stimulate primary responses. Because Mphi can be very abundant cells, especially at sites of infection and inflammation, they have the potential to play an important role in immune surveillance and the initiation of T cell immunity.     

Cross-priming as a predominant mechanism for inducing CD8(+) T cell responses in gene gun DNA immunization.

DNA immunization induces CD8(+) CTL responses by bone marrow-derived APCs, which are directly transfected with a plasmid DNA and/or acquire Ags from DNA-transfected non-APCs. To investigate the relative contribution of DNA-transfected APCs vs non-APCs to the initiation of CD8(+) T cell responses, we used tissue-specific promoter-directed gene expression and adoptive transfer systems in gene gun DNA immunization. In this study, we demonstrated that non-APC-specific gene expressions induced significant CD8(+) CTL and IFN-gamma-producing cells and Ab responses, whereas APC-specific gene expressions led to moderate CTL and IFN-gamma-producers, but no Ab responses. Interestingly, mice immunized with a non-APC-specific plasmid induced more rapid, vigorous, and prolonged proliferation of adoptively transferred Ag-specific CD8(+) T cells than APC-specific plasmid-immunized mice. In addition, the in vivo proliferative responses elicited by a non-APC-specific plasmid administration were dependent on TAP, but were independent of CD4(+) T cell help. Collectively, our results suggest that cross-priming, in which Ags expressed in non-APCs are taken up, processed, and presented by APCs, plays an important role in the initiation, magnitude, and maintenance of CD8(+) T cell responses in gene gun DNA immunization.     

Potential of transfected muscle cells to contribute to DNA vaccine immunogenicity

The mechanism(s) by which DNA vaccines trigger the activation of Ag-specific T cells is incompletely understood. A series of in vivo and in vitro experiments indicates plasmid transfection stimulates muscle cells to up-regulate expression of MHC class I and costimulatory molecules and to produce multiple cytokines and chemokines. Transfected muscle cells gain the ability to directly present Ag to CD8 T cells through an IFN-regulatory factor 3-dependent process. These findings suggest that transfected muscle cells at the site of DNA vaccination may contribute to the magnitude and/or duration of the immune response initiated by professional APCs.     

Effector CD4 cells are tolerized upon exposure to parenchymal self-antigen

It has long been established that exposure of naive T cells to specific Ag in the absence of adjuvant leads to tolerization. Nonetheless, the potential of effector CD4 cells to be tolerized has been less well characterized. To address this issue, we have used an adoptive transfer system in which naive TCR transgenic hemagglutinin (HA)-specific CD4(+) T cells are initially primed to express effector function upon exposure to an immunogenic recombinant vaccinia virus expressing HA, and then exposed to forms of HA that are tolerogenic for naive CD4 cells. HA-specific effector CD4 cells residing in both the spleen as well as in two separate nonlymphoid tissues were tolerized upon exposure to high doses of exogenous soluble HA peptide. Additionally, tolerance could also be induced by bone marrow-derived APCs that cross-present parenchymally derived self-HA. Thus, effector CD4 cells are susceptible to similar tolerogenic stimuli as are naive CD4 cells.     

Cutting edge: CD8+ effector T cells reject tumors by direct antigen recognition but indirect action on host cells

CD8(+) effector T cells recognize malignant cells by monitoring their surface for the presence of tumor-derived peptides bound to MHC class I molecules. In addition, tumor-derived Ags can be cross-presented to CD8(+) effector T cells by APCs. IFN-gamma production by CD8(+) T cells is often critical for tumor rejection. However, it remained unclear whether 1) CD8(+) T cells secrete IFN-gamma in response to Ag recognition on tumor cells or APCs and 2) whether IFN-gamma mediates its antitumor effect by acting on host or tumor cells. We show in this study that CD8(+) effector T cells can reject tumors in bone marrow-chimeric mice incapable of cross-presenting Ag by bone marrow-derived APCs and that tumor rejection required host cells to express IFN-gammaR. Together, CD8(+) effector T cells recognize Ag directly on tumor cells, and this recognition is sufficient to reject tumors by IFN-gamma acting on host cells.     

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.     

CD86 (B7-2) can function to drive MHC-restricted antigen-specific CTL responses in vivo

Activation of T cells requires both TCR-specific ligation by direct contact with peptide Ag-MHC complexes and coligation of the B7 family of ligands through CD28/CTLA-4 on the T cell surface. We recently reported that coadministration of CD86 cDNA along with DNA encoding HIV-1 Ags i.m. dramatically increased Ag-specific CTL responses. We investigated whether the bone marrow-derived professional APCs or muscle cells were responsible for the enhancement of CTL responses following CD86 coadministration. Accordingly, we analyzed CTL induction in bone marrow chimeras. These chimeras are capable of generating functional viral-specific CTLs against vaccinia virus and therefore represent a useful model system to study APC/T cell function in vivo. In vaccinated chimeras, we observed that only CD86 + Ag + MHC class I results in 1) detectable CTLs following in vitro restimulation, 2) detectable direct CTLs, 3) enhanced IFN-gamma production in an Ag-specific manner, and 4) dramatic tissue invasion of T cells. These results support that CD86 plays a central role in CTL induction in vivo, enabling non-bone marrow-derived cells to prime CTLs, a property previously associated solely with bone marrow-derived APCs.     

Comparison of filler DNA at immune, nonimmune, and oncogenic rearrangements suggests multiple mechanisms of formation.

Extra nucleotides (termed filler DNA) are commonly found at the junctions of genetic rearrangements in mammalian cells. The filler DNA at immune system rearrangements, which are called N regions, are generated at VDJ joints primarily by terminal deoxynucleotidyl transferase. However, the origin of filler DNA at genetic rearrangements in nonlymphoid cells is uncertain. In an analysis of more than 200 junctions that arose by circularization of transfected linear DNA (D. B. Roth and J. H. Wilson, Mol. Cell. Biol. 6:4295-4304, 1986), we found 18 junctions with extra nucleotides exactly at the point of circularization. Analysis of these 18 junctions indicated that nonlymphoid cells could add extra nucleotides to the ends of duplex DNA. The characteristics of the extra nucleotides at these junctions and at 31 other rearrangement junctions from nonlymphoid cells were quite similar, suggesting that many genetic rearrangements may pass through a stage with free DNA ends. A comparison of the filler DNA at these 49 nonimmune system rearrangements with 97 N regions derived from immune system rearrangements suggested that lymphoid and nonlymphoid cells use different mechanisms for insertion of filler DNA, as expected from the absence of detectable terminal deoxynucleotidyl transferase in cells from nonlymphoid tissues. The filler DNAs at a smaller group of 22 translocations associated with cancer had features in common with both immune and nonimmune system rearrangements and therefore may represent a mixture of these two processes. Mechanisms that might account for the presence of filler DNA in nonlymphoid cells are discussed.     

Priming of cytotoxic T lymphocytes by DNA vaccines: requirement for professional antigen presenting cells and evidence for antigen transfer from myocytes.

BACKGROUND: MHC class I molecule-restricted cytotoxic T-lymphocyte (CTL) responses are induced following either intramuscular (i.m.) injection of a DNA plasmid encoding influenza virus nucleoprotein (NP) or transplantation of myoblasts stably transfected with the NP gene, the latter indicating that synthesis of NP by myocytes in vivo is sufficient to induce CTL. The present study was designed to investigate the role of muscle cells and involvement of professional antigen-presenting cells (APCs) in priming CTL responses following DNA vaccination. MATERIALS AND METHODS: Parent-->F1 bone marrow (BM) chimeric mice were generated whose somatic cells include muscle cells bearing both parental MHC haplotypes, while their professional APCs express only the donor MHC haplotypes. RESULTS AND CONCLUSIONS: Upon injection of NP DNA, or after infection with influenza virus, CTL responses generated in the chimeras were restricted to the donor MHC haplotype. Thus cells of BM lineage were definitively shown to be responsible for priming such CTL responses after infection or DNA immunization. Moreover, expression of antigen by muscle cells in BM chimeric mice after myoblast transplantation is sufficient to induce CTL restricted only by the MHC haplotype of the donor BM. This indicates that transfer of antigen from myocytes to professional APCs can occur, thus obviating a requirement for direct transfection of BM-derived cells.     

Editing antigen presentation: antigen transfer between human B lymphocytes and macrophages mediated by class A scavenger receptors

B lymphocytes can function independently as efficient APCs. However, our previous studies demonstrate that both dendritic cells and macrophages are necessary to propagate immune responses initiated by B cell APCs. This finding led us to identify a process in mice whereby Ag-specific B cells transfer Ag to other APCs. In this study, we report the ability and mechanism by which human B lymphocytes can transfer BCR-captured Ag to macrophages. The transfer of Ag involves direct contact between the two cells followed by the capture of B cell-derived membrane and/or intracellular components by the macrophage. These events are abrogated by blocking scavenger receptor A, a receptor involved in the exchange of membrane between APCs. Macrophages acquire greater amounts of Ag in the presence of specific B cells than in their absence. This mechanism allows B cells to amplify or edit the immune response to specific Ag by transferring BCR-captured Ag to other professional APCs, thereby increasing the frequency of its presentation. Ag transfer may perpetuate chronic autoimmune responses to specific self-proteins and help explain the efficacy of B cell-directed therapies in human disease.     

TLR-activated dendritic cells enhance the response of aged naive CD4 T cells via an IL-6-dependent mechanism

The most effective immunological adjuvants contain microbial products, such as TLR agonists, which bind to conserved pathogen recognition receptors. These activate dendritic cells (DCs) to become highly effective APCs. We assessed whether TLR ligand-treated DCs can enhance the otherwise defective response of aged naive CD4 T cells. In vivo administration of CpG, polyinosinic-polycytidylic acid, and Pam(3)CSK(4) in combination with Ag resulted in the increased expression of costimulatory molecules and MHC class II by DCs, increased serum levels of the inflammatory cytokines IL-6 and RANTES, and increased cognate CD4 T cell responses in young and aged mice. We show that, in vitro, preactivation of DCs by TLR ligands makes them more efficient APCs for aged naive CD4 T cells. After T-DC interaction, there are enhanced production of inflammatory cytokines, particularly IL-6, and greater expansion of the aged T cells, resulting from increased proliferation and greater effector survival with increased levels of Bcl-2. TLR preactivation of both bone marrow-derived and ex vivo DCs improved responses. IL-6 produced by the activated DCs during cognate T cell interaction was necessary for enhanced aged CD4 T cell expansion and survival. These studies suggest that some age-associated immune defects may be overcome by targeted activation of APCs by TLR ligands.     

Modulation of proinflammatory responses to Pneumocystis carinii f. sp. muris in neonatal mice by granulocyte-macrophage colony-stimulating factor and IL-4: role of APCs

Clearance of Pneumocystis carinii f. sp. muris (PC) organisms from the lungs of neonatal mice is delayed due to failure of initiation of inflammation over the first 3 wk after infection. The ability of neonatal lung CD11c(+) dendritic cells (DCs) to induce Ag-specific T cell proliferative responses was significantly reduced compared with adult lung DCs. However, neonatal bone marrow-derived DCs were as competent at presenting PC Ag as were adult bone marrow-derived DCs. Because GM-CSF mRNA expression and activity were significantly reduced in neonatal lungs compared with adults, we treated neonates with exogenous GM-CSF and IL-4 and found enhanced clearance of PC compared with untreated neonates. This was associated with increased lung TNF-alpha, IL-12p35, and IL-18 mRNA expression, indicating enhanced innate immune responses. Cytokine-treated mice had marked expansion of CD11c(+) DCs with up-regulated MHC-II in the lungs. Moreover, increased numbers of activated CD4(+)CD44(high)CD62L(low) cells in the lungs and draining lymph nodes suggested improved Ag presentation by the APCs. Together these data indicate that neonatal lungs lack maturation factors for efficient cellular functioning, including APC maturation.     

Expression of antigen on mature lymphocytes is required to induce T cell tolerance by gene therapy

Expression of a retrovirally encoded allogeneic MHC class I gene in bone marrow-derived cells can be used to induce tolerance to the product of the retrovirally transduced gene. In this work we examined whether expression of a retrovirally transduced allogeneic MHC class I gene in bone marrow-derived cells from recombinase-activating gene-1 (RAG-1)-deficient mice was sufficient to induce tolerance when transplanted into conditioned hosts together with bone marrow from MHC-matched wild-type mice. Reconstitution of mice with either MHC-matched RAG-1-deficient or wild-type bone marrow transduced with the allogeneic MHC class I gene H-2K(b) led to long-term expression of K(b) on the surface of bone marrow-derived hematopoietic lineages. T cells from mice reconstituted with H-2K(b)-transduced wild-type bone marrow were tolerant to K(b). In contrast, expression of K(b) in the periphery of mice reconstituted with a mixture of retrovirally transduced RAG-1-deficient bone marrow and mock-transduced wild-type bone marrow fell below detectable levels by 4 wk after transplantation. T cells that developed in these mice appeared to be hyporesponsive to K(b), demonstrating that expression of K(b) on bone marrow-derived APCs was not sufficient to induce tolerance. Our data suggest that induction of tolerance in molecular chimeras requires expression of the retrovirally transduced allogeneic MHC Ag on the surface of mature lymphocytes that populate the host thymus.     

Enhancement of Sindbis virus self-replicating RNA vaccine potency by linkage of Mycobacterium tuberculosis heat shock protein 70 gene to an antigen gene

Recently, self-replicating RNA vaccines (RNA replicons) have emerged as an effective strategy for nucleic acid vaccine development. Unlike naked DNA vaccines, RNA replicons eventually cause lysis of transfected cells and therefore do not raise the concern of integration into the host genome. We evaluated the effect of linking human papillomavirus type 16 E7 as a model Ag to Mycobacterium tuberculosis heat shock protein 70 (HSP70) on the potency of Ag-specific immunity generated by a Sindbis virus self-replicating RNA vector, SINrep5. Our results indicated that this RNA replicon vaccine containing an E7/HSP70 fusion gene generated significantly higher E7-specific T cell-mediated immune responses in vaccinated mice than did vaccines containing the wild-type E7 gene. Furthermore, our in vitro studies demonstrated that E7 Ag from E7/HSP70 RNA replicon-transfected cells can be processed by bone marrow-derived dendritic cells and presented more efficiently through the MHC class I pathway than can wild-type E7 RNA replicon-transfected cells. More importantly, the fusion of HSP70 to E7 converted a less effective vaccine into one with significant potency against E7-expressing tumors. This antitumor effect was dependent on NK cells and CD8(+) T cells. These results indicated that fusion of HSP70 to an Ag gene may greatly enhance the potency of self-replicating RNA vaccines.     

Amiloride enhances antigen specific CTL by faciliting HBV DNA vaccine entry into cells

The induction of relatively weak immunity by DNA vaccines in humans can be largely attributed to the low efficiency of transduction of somatic cells. Although formulation with liposomes has been shown to enhance DNA transduction of cultured cells, little, if any, effect is observed on the transduction of somatic tissues and cells. To improve the rate of transduction, DNA vaccine delivery by gene gun and the recently developed electroporation techniques have been employed. We report here that to circumvent requirement for such equipment, amiloride, a drug that is prescribed for hypertension treatment, can accelerate plasmid entry into antigen presenting cells (APCs) both in vitro and in vivo. The combination induced APCs more dramatically in both maturation and cytokine secretion. Amiloride enhanced development of full CD8 cytolytic function including induction of high levels of antigen specific CTL and expression of IFN-γ+perforin+granzymeB+ in CD8+ T cells. Thus, amiloride is a facilitator for DNA transduction into host cells which in turn enhances the efficiency of the immune responses.     

Extracellular Mg concentration and Ca blockers modulate the initial steps of the response of Th2 lymphocytes in co-culture with macrophages and dendritic cells

Magnesium is highly involved in the metabolic network such that even subtle disturbances in its homeostasis affect many cellular functions, including calcium homeostasis, signal transduction, energy metabolism, membrane stability and cell proliferation. Recently, magnesium level has been proposed to modulate the priming and activity of immune cells. We studied the behavior of antigen-presenting cells (APCs) and T lymphocytes after altering the magnesium/calcium balance. We used two different populations of primary APCs, i.e. bone marrowderived dendritic cells and bone marrow-derived macrophages, while D10.G4.1 cells served as a model of responding Th2 cells. Our principal findings are the following: (i) the extracellular magnesium concentration had no significant impact on endocytosis by bone marrow-derived APCs, (ii) high concentrations of extracellular magnesium, with or without calcium antagonists, significantly decreased IL-4 and IL-10 secretion by Th2 cells in a co-culture system of APCsandTh2lymphocytes, (iii) proliferation ofTh2cells in co-culture systems was significantly inhibited by calcium antagonists independently from extracellular magnesium concentrations. Our results suggest that alterations of magnesium and calcium homeostasis impact on some crucial steps of the immune response.     

Cutting edge: Nicastrin and related components of γ-secretase generate a peptide epitope facilitating immune recognition of intracellular mycobacteria, through MHC class II-dependent priming of T cells

Bacillus Calmette-Guérin (BCG), the antituberculosis vaccine, localizes within immature phagosomes of macrophages and dendritic cells (APCs), and avoids lysosomal degradation. BCG-derived antigenic peptides are thus inefficiently processed by APCs, and we investigated alternate mechanisms of Ag processing. Proteomics identified that BCG phagosomes are enriched for nicastrin, APH, and presenilin components of γ-secretase, a multimeric protease. Using an in vitro Ag presentation assay and BCG-infected APCs, we found γ-secretase components to cleave BCG-derived Ag85B to produce a peptide epitope, which, in turn, primed IL-2 release from Ag85B-specific T cell hybridoma. siRNA knockdown or chemical inhibition of γ-secretase components using L685458 decreased the ability of BCG or Mycobacterium tuberculosis-infected APCs to present Ag85B. In addition, L685485 inhibition of γ-secretase led to a decreased ability of BCG-dendritic cells to immunize mice and induce Ag85B-specific CD4 T cells in vivo. Because BCG and M. tuberculosis sequester within APCs preventing immune recognition, γ-secretase components appear to fortuitously process the immunodominant Ag85B, facilitating immune recognition.     

Transfection of immature murine bone marrow-derived dendritic cells with the granulocyte-macrophage colony-stimulating factor gene potently enhances their in vivo antigen-presenting capacity.

Ag presentation by dendritic cells (DC) is crucial for induction of primary T cell-mediated immune responses in vivo. Because DC culture from blood or bone marrow-derived progenitors is now clinically applicable, this study investigated the effectiveness of in vitro-generated murine bone marrow-derived DC (Bm-DC) for in vivo immunization protocols. Previous studies demonstrated that GM-CSF is an essential growth and differentiation factor for DC in culture and that in vivo administration of GM-CSF augments primary immune responses, which renders GM-CSF an attractive candidate to further enhance the effectiveness of DC-based immunotherapy protocols. Therefore, immature Bm-DC were transiently transfected with the GM-CSF gene and tested for differentiation, migration, and Ag-presenting capacity in vitro and in vivo. In vitro, GM-CSF gene-transfected Bm-DC were largely unaltered with regard to MHC and costimulatory molecule expression as well as alloantigen or peptide Ag-presenting capacity. When used for in vivo immunizations, however, the Ag-presenting capacity of GM-CSF gene-transfected Bm-DC was greatly enhanced compared with mock-transfected or untransfected cells, as determined by their effectiveness to induce primary immune reactions against hapten, protein Ag, and tumor Ag, respectively. Increased effectiveness in vivo correlated with the better migratory capacity of GM-CSF gene-transfected Bm-DC. These results show that GM-CSF gene transfection significantly enhances the capacity of DC to induce primary immune responses in vivo, which might also improve DC-based vaccines currently under clinical investigation.     

Antigen recognition is facilitated by invadosome-like protrusions formed by memory/effector T cells

Adaptive immunity requires that T cells efficiently scan diverse cell surfaces to identify cognate Ag. However, the basic cellular mechanisms remain unclear. In this study, we investigated this process using vascular endothelial cells, APCs that possess a unique and extremely advantageous, planar morphology. High-resolution imaging revealed that CD4 memory/effector T cells dynamically probe the endothelium by extending submicron-scale, actin-rich "invadosome/podosome-like protrusions" (ILPs). The intimate intercellular contacts enforced by ILPs consistently preceded and supported T cell activation in response to endothelial MHC class II/Ag. The resulting calcium flux stabilized dense arrays of ILPs (each enriched in TCR, protein kinase C-θ, ZAP70, phosphotyrosine, and HS1), forming what we term a podo-synapse. Similar findings were made using CD8 CTLs on endothelium. Furthermore, careful re-examination of both traditional APC models and professional APCs suggests broad relevance for ILPs in facilitating Ag recognition. Together, our results indicate that ILPs function as sensory organelles that serve as actuators of immune surveillance.