Recent evidence suggests that in addition to their well known stimulatory properties, dendritic cells (DCs) may play a major role in peripheral tolerance. It is still unclear whether a distinct subtype or activation status of DC exists that promotes the differentiation of suppressor rather than effector T cells from naive precursors. In this work, we tested whether the naturally occurring CD4+ CD25+ regulatory T cells (Treg) may control immune responses induced by DCs in vivo. We characterized the immune response induced by adoptive transfer of antigen-pulsed mature DCs into mice depleted or not of CD25+ cells. We found that the development of major histocompatibility complex class I and II–restricted interferon γ–producing cells was consistently enhanced in the absence of Treg. By contrast, T helper cell (Th)2 priming was down-regulated in the same conditions. This regulation was independent of interleukin 10 production by DCs. Of note, splenic DCs incubated in vitro with Toll-like receptor ligands (lipopolysaccharide or CpG) activated immune responses that remained sensitive to Treg function. Our data further show that mature DCs induced higher cytotoxic activity in CD25-depleted recipients as compared with untreated hosts. We conclude that Treg naturally exert a negative feedback mechanism on Th1-type responses induced by mature DCs in vivo.
primary response; T helper cell type 1/type 2 balance; regulation; inflammation; Toll-like receptors
Cells of the Lyl subclass generate helper activity in both primary and secondary responses to sheep erythrocytes (SRBC). In contrast, after priming with SRBC, cells of the Ly-2+ subclasses, in particular Ly23 cells, have suppressive activity. The degree of Ly23-mediated suppression is directly proportional to the amount of antigen (SRBC) used for priming. Suppression by Ly23 cells is specific, because Ly23 cells from SRBC-primed animals do not suppress the response to horse erythrocytes, and vice versa. Thus, both cytotoxic and specific suppressor functions are mediated by T cells of a subclass, provisionally designated TCS, which can be distinguished from helper T cells (TH), by their Ly phenotypes. It remains to be determined whether killing and suppression are functionally interrelated properties of a single Ly23 subclass, or whether the Ly23 population comprises two subclasses whose surface phenotypes are not yet distinguishable by immunogenetic criteria.
Using granulocyte/macrophage colony-stimulating factor (GM-CSF) and interleukin 4 we have established dendritic cell (DC) lines from blood mononuclear cells that maintain the antigen capturing and processing capacity characteristic of immature dendritic cells in vivo. These cells have typical dendritic morphology, express high levels of major histocompatibility complex (MHC) class I and class II molecules, CD1, Fc gamma RII, CD40, B7, CD44, and ICAM-1, and lack CD14. Cultured DCs are highly stimulatory in mixed leukocyte reaction (MLR) and are also capable of triggering cord blood naive T cells. Most strikingly, these DCs are as efficient as antigen-specific B cells in presenting tetanus toxoid (TT) to specific T cell clones. Their efficiency of antigen presentation can be further enhanced by specific antibodies via FcR- mediated antigen uptake. Incubation of these cultured DCs with tumor necrosis factor alpha (TNF-alpha) or soluble CD40 ligand (CD40L) for 24 h results in an increased surface expression of MHC class I and class II molecules, B7, and ICAM-1 and in the appearance of the CD44 exon 9 splice variant (CD44-v9); by contrast, Fc gamma RII is markedly and sometimes completely downregulated. The functional consequences of the short contact with TNF-alpha are in increased T cell stimulatory capacity in MLR, but a 10-fold decrease in presentation of soluble TT and a 100-fold decrease in presentation of TT-immunoglobulin G complexes.
Two subsets of murine splenic dendritic cells, derived from distinct precursors, can be distinguished by surface expression of CD8α homodimers. The functions of the two subsets remain controversial, although it has been suggested that the lymphoid-derived (CD8α+) subset induces tolerance, whereas the myeloid-derived (CD8α−) subset has been shown to prime naive T cells and to generate memory responses. To study their capacity to prime or tolerize naive CD4+ T cells in vivo, purified CD8α+ or CD8α− dendritic cells were injected subcutaneously into normal mice. In contrast to CD8α− dendritic cells, the CD8α+ fraction failed to traffic to the draining lymph node and did not generate responses to intravenous peptide. However, after in vitro pulsing with peptide, strong in vivo T cell responses to purified CD8α+ dendritic cells could be detected. Such responses may have been initiated via transfer of peptide–major histocompatibility complex complexes to migratory host CD8α− dendritic cells after injection. These data suggest that correlation of T helper cell type 1 (Th1) and Th2 priming with injection of CD8α+ and CD8α− dendritic cells, respectively, may not result from direct T cell activation by lymphoid versus myeloid dendritic cells, but rather from indirect modification of the response to immunogenic CD8α− dendritic cells by CD8α+ dendritic cells.
dendritic cell; T cell; antigen presentation; tolerance
Dendritic cells (DCs) play an important role in CD4+ T helper (Th) cell differentiation and in the initiation of both protective and pathogenic immunity. Granulocyte/macrophage colony-stimulating factor (GM-CSF) is a DC growth factor critical for the induction of experimental autoimmune encephalomyelitis (EAE) and other autoimmune diseases, yet its mechanism of action in vivo is not fully defined. We show that GM-CSF is directly required for the accumulation of radiosensitive dermal-derived langerin+CD103+ DCs in the skin and peripheral lymph nodes under steady-state and inflammatory conditions. Langerin+CD103+ DCs stimulated naive myelin-reactive T cells to proliferate and produce IFN-γ and IL-17. They were superior to other DC subsets in inducing expression of T-bet and promoting Th1 cell differentiation. Ablation of this subset in vivo conferred resistance to EAE. The current report reveals a previously unidentified role for GM-CSF in DC ontogeny and identifies langerin+CD103+ DCs as an important subset in CD4+ T cell–mediated autoimmune disease.
Concanavalin A, a nonspecific polyclonal activator of T lymphocytes, activates Lyl and Ly23 subclasses to the same degree. After activation, the Ly23 subclass, but not the Lyl subclass, has the following properties: (a) Suppression of the antibody response to sheep erythrocytes (SRBC) in vitro. (b) Production of a soluble factor that suppresses the anti-SRBC response in vitro. (c) Suppression of the generation of cell-mediated cytotoxicity to H-2 target cells in vitro. Con A-activated cells of the Lyl subclass, but not the Ly23 subclass, express helper function in the anti-SRBC response in vitro. Because the intact Con A-stimulated T-cell population contains both cell types, these cells do not exert detectable helper effects in an anti-SRBC system in vitro, because the helper effect of Lyl cells is masked by the suppressor effect of the Ly23 cells. Each function is revealed by eliminating one or the other population with the relevant Ly antiserum. The resting T-cell population, before activation by Con A, also contains already programmed Lyl and Ly23 cells with similar helper and suppressor potentials, respectively. This is revealed by experiments with Ly subclasses which have been separated from the resting T-cell population and then stimulated by Con A. Thus helper and suppressor functions, as expressed in these systems, are manifestations of separate T-cell-differentiative pathways and do not depend upon stimulation of the cells by antigen.
Because of different cytokine responsiveness, surface receptor, and transcription factor expression, human CD11c− natural type I interferon–producing cells (IPCs) and CD11c+ dendritic cells were thought to derive through lymphoid and myeloid hematopoietic developmental pathways, respectively. To directly test this hypothesis, we used an in vitro assay allowing simultaneous IPC, dendritic cell, and B cell development and we tested lymphoid and myeloid committed hematopoietic progenitor cells for their developmental capacity. Lymphoid and common myeloid and granulocyte/macrophage progenitors were capable of developing into both functional IPCs, expressing gene transcripts thought to be associated with lymphoid lineage development, and into dendritic cells. However, clonal progenitors for both populations were about fivefold more frequent within myeloid committed progenitor cells. Thus, in humans as in mice, natural IPC and dendritic cell development robustly segregates with myeloid differentiation. This would fit with natural interferon type I–producing cell and dendritic cell activity in innate immunity, the evolutionary older arm of the cellular immune system.
human hematopoietic progenitors; plasmacytoid dendritic cells; dendritic cells; development; hematopoietic lineage commitment
Distinct dendritic cell (DC) subsets have been suggested to be preprogrammed to direct either T helper cell (Th) type 1 or Th2 development, although more recently different pathogen products or stimuli have been shown to render these DCs more flexible. It is still unclear how distinct mouse DC subsets cultured from bone marrow precursors, blood, or their lymphoid tissue counterparts direct Th differentiation. We show that mouse myeloid and plasmacytoid precursor DCs (pDCs) cultured from bone marrow precursors and ex vivo splenic DC subsets can induce the development of both Th1 and Th2 effector cells depending on the dose of antigen. In general, high antigen doses induced Th1 cell development whereas low antigen doses induced Th2 cell development. Both cultured and ex vivo splenic plasmacytoid-derived DCs enhanced CD4+ T cell proliferation and induced strong Th1 cell development when activated with the Toll-like receptor (TLR)9 ligand CpG, and not with the TLR4 ligand lipopolysaccharide (LPS). The responsiveness of plasmacytoid pDCs to CpG correlated with high TLR9 expression similarly to human plasmacytoid pDCs. Conversely, myeloid DCs generated with granulocyte/macrophage colony-stimulating factor enhanced Th1 cell development when stimulated with LPS as a result of their high level of TLR4 expression. Polarized Th1 responses resulting from high antigen dose were not additionally enhanced by stimulation of DCs by TLR ligands. Thus, the net effect of antigen dose, the state of maturation of the DCs together with the stimulation of DCs by pathogen-derived products, will determine whether a Th1 or Th2 response develops.
dendritic cell; Th1; Th2; TLR; cytokines
Soluble egg antigens of the parasitic helminth Schistosoma mansoni (S. mansoni egg antigen [SEA]) induce strong Th2 responses both in vitro and in vivo. However, the specific molecules that prime the development of Th2 responses have not been identified. We report that omega-1, a glycoprotein which is secreted from S. mansoni eggs and present in SEA, is capable of conditioning human monocyte-derived dendritic cells in vitro to drive T helper 2 (Th2) polarization with similar characteristics as whole SEA. Furthermore, using IL-4 dual reporter mice, we show that both natural and recombinant omega-1 alone are sufficient to generate Th2 responses in vivo, even in the absence of IL-4R signaling. Finally, omega-1–depleted SEA displays an impaired capacity for Th2 priming in vitro, but not in vivo, suggesting the existence of additional factors within SEA that can compensate for the omega-1–mediated effects. Collectively, we identify omega-1, a single component of SEA, as a potent inducer of Th2 responses.
T cells recognize peptides that are bound to MHC molecules on the surface of different types of antigen-presenting cells (APC). Antigen presentation most often is studied using T cells that have undergone priming in situ, or cell lines that have been chronically stimulated in vitro. The use of primed cells provides sufficient numbers of antigen- reactive lymphocytes for experimental study. A more complete understanding of immunogenicity, however, requires that one develop systems for studying the onset of a T cell response from unprimed lymphocytes, especially in situ. Here it is shown that mouse T cells can be reliably primed in situ using dendritic cells as APC. The dendritic cells were isolated from spleen, pulsed with protein antigens, and then administered to naive mice. Antigen-responsive T cells developed in the draining lymphoid tissue, and these T cells only recognized protein when presented on cells bearing the same MHC products as the original priming dendritic cells. In contrast, little or no priming was seen if antigen-pulsed spleen cells or peritoneal cells were injected. Since very small amounts of the foreign protein were visualized within endocytic vacuoles of antigen-pulsed dendritic cells, it is suggested that dendritic cells have a small but relevant vacuolar system for presenting antigens over a several day period in situ.
Borrelia burgdorferi-pulsed dendritic cells and epidermal cells were able to initiate the production of anti-outer surface protein A (OspA) antibody in vitro with normal T and B cells from either BALB/c or C3H/HeJ mice. Inhibition of anti-B. burgdorferi antibody production was observed after 3 days, but not after 2 days, of exposure of the antigen-presenting cells to tumor necrosis factor alpha +/- granulocyte-macrophage colony-stimulating factor. Furthermore, splenic dendritic cells pulsed in vitro with live B. burgdorferi spirochetes and then adoptively transferred into naive syngeneic mice mediated a protective immune response against tick-transmitted spirochetes. This protection appeared not to be due to killing of spirochetes in the feeding ticks, since ticks fed to repletion on B. burgdorferi-pulsed dendritic cell-sensitized mice still harbored live spirochetes. Western blot analysis of the sera collected from dendritic cell-sensitized mice demonstrated that the mice responded to a limited set of B. burgdorferi antigens, including OspA, -B, and -C compared to control groups that either had received unpulsed dendritic cells or were not treated. Finally, mice in the early stage of B. burgdorferi infection were able to develop anti-OspA antibody following injection with B. burgdorferi-pulsed dendritic cells. Our results demonstrate for the first time that adoptive transfer of B. burgdorferi-pulsed dendritic cells induces a protective immune response against tick-transmitted B. burgdorferi and stimulates the production of antibodies specific for a limited set of B. burgdorferi antigens in vivo.
Multilineage donor-derived hematopoietic cell chimerism is a persistent feature of spontaneously tolerant mouse liver allograft recipients. We have shown previously that normal liver-derived precursors of “chimeric” dendritic cells (DC) propagated in vitro migrate in vivo to T-dependent areas of allogeneic lymphoid tissue, where they or their progeny appear to persist indefinitely. In this study, granulocyte-macrophage colony-stimulating factor (GM-CSF)+interleukin-4 (IL-4) were used to propagate DC progenitors from freshly isolated mouse bone marrow. The progenitor cells gave rise in 7–10 days to potent antigen-presenting cells (APC) that stimulated naive allogeneic T cells in primary mixed leukocyte cultures (MLC). The culture method, together with the reverse transcriptase-polymerase chain reaction (RT-PCR) for the detection of donor and recipient strain major histocompatibility complex (MHC) class II mRNA was used to test whether donor-derived DC could be propagated from the bone marrow of unmodified, orthotopic liver allograft recipients. Freshly isolated bone marrow from these transplanted animals contained small numbers of donor cells and responded to GM-CSF+IL-4 stimulation. In addition to cells expressing recipient (B10) phenotype (H-2Kb+; Iab+), a minor population of donor (B10.BR)-derived cells (H-2Kk+; Iak) were also propagated from liver graft recipients euthanized two weeks posttransplant. DC sorted from these cultures exhibited stimulatory activity for recipient strain T cells consistent with a low level (<1%) of donor DC propagation. The immunologic role of donor-derived DC progenitors in liver allograft recipients and its relation to the induction and maintenance of donor-specific unresponsiveness remains to be determined.
Dendritic cells (DCs) are professional antigen-presenting cells that can prime T cells and polarize the cellular immune response. Because Th1-type immune responses have been connected to success in combating viral infection, a promising therapeutic application of DCs would be their differentiation in vitro and injection back into the host to boost an immune response in infected animals. This study was aimed both at developing a protocol to cultivate feline DCs in the absence of exogenous proteins for their use in vivo and at investigating what might be the most appropriate stimulus to induce their maturation in vitro and finding correlates of maturation. We generated DCs from peripheral blood monocytes in the presence of feline interleukin-4 and granulocyte-macrophage colony stimulating factor, and after 5 days their maturation was induced with either lipopolysaccharide, human recombinant tumor necrosis factor alpha, poly(I:C), or activated feline platelets. After 48 h, their CD14, CD1a, major histocompatibility complex class II, and B7.1 surface expression was analyzed in parallel with their ability to uptake antigen or prime a mixed leukocyte reaction. The results presented show that feline DCs cultured in autologous plasma differentiate and are able to mature in the presence of stimuli similar to the ones currently used for other species. The present work sets the grounds for future use of DCs obtained by the protocol described for in vivo vaccination and immunotherapy of feline immunodeficiency virus-infected cats.
DCs (dendritic cells) function as sentinels of the immune system. They traffic from the blood to the tissues where, while immature, they capture antigens. They then leave the tissues and move to the draining lymphoid organs where, converted into mature DC, they prime naive T cells. This suggestive link between DC traffic pattern and functions led us to investigate the chemokine responsiveness of DCs during their development and maturation. DCs were differentiated either from CD34+ hematopoietic progenitor cells (HPCs) cultured with granulocyte/macrophage colony–stimulating factor (GM-CSF) plus tumor necrosis factor (TNF)-α or from monocytes cultured with GM-CSF plus interleukin 4. Immature DCs derived from CD34+ HPCs migrate most vigorously in response to macrophage inflammatory protein (MIP)-3α, but also to MIP-1α and RANTES (regulated on activation, normal T cell expressed and secreted). Upon maturation, induced by either TNF-α, lipopolysaccharide, or CD40L, DCs lose their response to these three chemokines when they acquire a sustained responsiveness to a single other chemokine, MIP-3β. CC chemokine receptor (CCR)6 and CCR7 are the only known receptors for MIP-3α and MIP-3β, respectively. The observation that CCR6 mRNA expression decreases progressively as DCs mature, whereas CCR7 mRNA expression is sharply upregulated, provides a likely explanation for the changes in chemokine responsiveness. Similarly, MIP-3β responsiveness and CCR7 expression are induced upon maturation of monocyte- derived DCs. Furthermore, the chemotactic response to MIP-3β is also acquired by CD11c+ DCs isolated from blood after spontaneous maturation. Finally, detection by in situ hybridization of MIP-3α mRNA only within inflamed epithelial crypts of tonsils, and of MIP-3β mRNA specifically in T cell–rich areas, suggests a role for MIP-3α/CCR6 in recruitment of immature DCs at site of injury and for MIP-3β/CCR7 in accumulation of antigen-loaded mature DCs in T cell–rich areas.
dendritic cells; chemokines; migration; maturation; regulation; in vivo expression
Immunization with dendritic cells pulsed ex vivo with antigens has been successfully used to elicit primary antigen-specific immune responses. We report that mouse bone marrow-derived dendritic cells pulsed with inactivated chlamydial organisms induced strong protection against live chlamydial infection in a mouse lung infection model. Either the dendritic cells or chlamydial organisms alone or macrophages similarly pulsed with chlamydial organisms failed to induce any significant protection. These observations suggest that dendritic cells can efficiently process and present chlamydial antigens to naive T cells in vivo. Mice immunized with the chlamydia-pulsed dendritic cells preferentially developed a Th1 cell-dominant response while mice immunized with the other immunogens did not, suggesting a correlation between a Th1 cell-dominant response and protection against chlamydial infection. We further found that dendritic cells produced a large amount of interleukin 12 (IL-12) upon ex vivo pulsing with inactivated chlamydial organisms, which may allow the dendritic cells to direct a Th1 cell-dominant response. Dendritic cells from mice deficient in the IL-12 p40 gene failed to produce IL-12 after a similar ex vivo pulse with chlamydial organisms, and more importantly, immunization with these dendritic cells failed to induce a Th1 cell-dominant response and did not induce strong protection against chlamydial infection. Thus, the ability of dendritic cells to efficiently process and present chlamydial antigens and to produce IL-12 upon chlamydial-organism stimulation are both required for the induction of protection against chlamydial infection. This information may be useful for the further design of effective chlamydial vaccines.
Dendritic cell (DC)-based immunization is a potent strategy to direct prompt and durable immune responses against viral reactivations after transplantations. Here, we show that overnight lentiviral vector (LV) gene transfer into human monocytes co-expressing granulocyte-macrophage colony stimulating factor and interleukin (IL)-4 induced self-differentiated DCs (SMART-DCs) with stable DC immunophenotype over weeks in culture and secreted several inflammatory cytokines. SMART-DCs injected subcutaneously in immunodeficient NOD.Rag1−/−.IL2rγ−/− (NRG) mice 1 day after LV transduction were stable for a month in vivo. “Conventional” DCs (cDCs) and SMART-DCs were compared with regard to their potency to accelerate the expansion, biodistribution, and antigenic stimulation of autologous human T cells. Peripheral blood cells obtained from human cytomegalovirus (hCMV)-reactive donors and full-length hCMV pp65 antigenic protein or peptides were used. DCs loaded with pp65 were administered subcutaneously into NRG mice as a preconditioning treatment a week prior to intravenous infusion with T cells. Optical imaging analyses demonstrated that in mice preconditioned with SMART-DC-pp65, T cells were directly recruited to the immunization site and subsequently spread to the spleen and other organs. A dramatic expansion of both human CD8+ and CD4+ T cells could be observed within a few days after infusion, and this was associated with consistent measurable CD8+ effector memory T-cell responses against different pp65 epitopes. Thus, this mouse model demonstrates the proof-of-principle for SMART-DCs to accelerate expansion of human lymphocytes, resulting in poly-functional and antigen-specific immune responses against hCMV-pp65.
Salguero and colleagues show proof of concept that preconditioning hosts with lentiviral vector-programmed dendritic cells expressing a viral antigen before T cell infusion accelerates T cell engraftment, biodistribution, and stimulation of antigen-specific immune responses in permissive mice.
Protective immunity at the gut-associated mucosal tissue is induced primarily by oral/rectal immunization owing to the need for targeting antigen to the gut-resident dendritic cells (DC). Here we show that an adenovirus type 5 (Ad5) based HIV-1 vaccine can prime a durable antigen-specific CD8 T cell response in the gut following intramuscular immunization in mice. The ability of Ad5 to prime gut homing CD8 T cells in vivo was associated with Ad5-induced expression of retinal dehydrogenase (RALDH) enzymes in conventional DC. The Ad5-mediated induction of RALDH did not require signaling through toll-like receptors, DNA-dependent activator of IRFs and several MAP kinases, or replication capacity of the virus, but was dependant on NF-κB and granulocyte-macrophage colony-stimulating factor. These results provide an innate mechanism through which Ad5-stimulated DC prime gut homing CD8 T cells and have implications for the development of novel mucosal adjuvants for subunit vaccines administered via the intramuscular route.
The early induction of interleukin (IL)-12 is a critical event in determining the development of both innate resistance and adaptive immunity to many intracellular pathogens. Previous in vitro studies have suggested that the macrophage (MΦ) is a major source of the initial IL-12 produced upon microbial stimulation and that this response promotes the differentiation of protective T helper cell 1 (Th1) CD4+ lymphocytes from precursors that are primed on antigen-bearing dendritic cells (DC). Here, we demonstrate by immunolocalization experiments and flow cytometric analysis that, contrary to expectation, DC and not MΦ are the initial cells to synthesize IL-12 in the spleens of mice exposed in vivo to an extract of Toxoplasma gondii or to lipopolysaccharide, two well characterized microbial stimulants of the cytokine. Importantly, this production of IL-12 occurs very rapidly and is independent of interferon γ priming or of signals from T cells, such as CD40 ligand. IL-12 production by splenic DC is accompanied by an increase in number of DCs, as well as a redistribution to the T cell areas and the acquisition of markers characteristic of interdigitating dendritic cells. The capacity of splenic DC but not MΦ to synthesize de novo high levels of IL-12 within hours of exposure to microbial products in vivo, as well as the ability of the same stimuli to induce migration of DC to the T cell areas, argues that DC function simultaneously as both antigen-presenting cells and IL-12 producing accessory cells in the initiation of cell-mediated immunity to intracellular pathogens. This model avoids the need to invoke a three-cell interaction for Th1 differentiation and points to the DC as both a sentinel for innate recognition and the dictator of class selection in the subsequent adaptive response.
Mature dendritic cells (DCs) are powerful antigen presenting cells that have the unique capacity to migrate to the T cell zone of draining lymph nodes after subcutaneous injection. Here we report that treatment of antigen-pulsed mature DCs with tumor necrosis factor (TNF)-related activation-induced cytokine (TRANCE), a TNF family member, before immunization enhances their adjuvant capacity and elicits improved T cell priming in vivo, such that both primary and memory T cell immune responses are enhanced. By enumerating migratory DCs in the draining lymph nodes and by studying their function in stimulating naive T cells, we show that one of the underlying mechanisms for enhanced T cell responses is an increase in the number of ex vivo antigen-pulsed DCs that are found in the T cell areas of lymph nodes. These results suggest that the longevity and abundance of mature DCs at the site of T cell priming influence the strength of the DC-initiated T cell immunity in situ. Our findings have the potential to improve DC-based immunotherapy; i.e., the active immunization of humans with autologous DCs that have been pulsed with clinically significant antigens ex vivo.
TRANCE; dendritic cells; T cell; immunization
Resistance to several prevalent infectious diseases requires both cellular and humoral immune responses. T cell immunity is initiated by mature dendritic cells (DCs) in lymphoid organs, whereas humoral responses to most antigens require further collaboration between primed, antigen-specific helper T cells and naive or memory B cells. To determine whether antigens delivered to DCs in lymphoid organs induce T cell help for antibody responses, we targeted a carrier protein, ovalbumin (OVA), to DCs in the presence of a maturation stimulus and assayed for antibodies to a hapten, (4-hydroxy-3-nitrophenyl) acetyl (NP), after boosting with OVA-NP. A single DC-targeted immunization elicited long-lived T cell helper responses to the carrier protein, leading to large numbers of antibody-secreting cells and high titers of high-affinity antihapten immunoglobulin Gs. Small doses of DC-targeted OVA induced higher titers and a broader spectrum of anti-NP antibody isotypes than large doses of OVA in alum adjuvant. Similar results were obtained when the circumsporozoite protein of Plasmodium yoelii was delivered to DCs. We conclude that antigen targeting to DCs combined with a maturation stimulus produces broad-based and long-lived T cell help for humoral immune responses.
The aim of this study was to develop an immunization procedure avoiding external adjuvant. Data are presented showing that syngeneic dendritic cells (DC), which have been pulsed in vitro with antigen, induce a strong antibody response in mice. By contrast, antigen (Ag)-pulsed low- density B cells, although equally able to induce interleukin 2 secretion by an Ag-specific T cell hybridoma in vitro, only weakly prime the mice in vivo. Moreover, we show that the injection of Ag- pulsed DC induces the synthesis of isotypes similar to the immunoglobulin classes detected after immunization with the same Ag in complete Freund's adjuvant. Importantly, high amounts of IgG2a antibodies are produced, suggesting that T helper type 1 cells are activated. Collectively, these data indicate that DC can initiate a primary humoral response and that they may be used as physiological adjuvant in vivo.
We present evidence for intrathymic presentation of soluble circulating antigens in vivo. Our results show that proteins of different molecular weight enter the mouse thymus rapidly after i.v. injection. The intrathymic presence of antigen was assayed by proliferation of cloned antigen-specific T helper cells, which were cocultured with purified thymic stromal cells; stromal cells were isolated and purified as lymphostromal cell complexes, which preexist in vivo. Antigen presentation copurified with non-adherent medullary dendritic cells (DC) (interdigitating cells). I-A- cortical macrophages forming thymocyte rosettes in vivo and I-A+ cortical epithelial cells forming thymic nurse cells (TNC) in vivo did not act as antigen presenting cells (APC) after antigen pulsing in vivo or in vitro. Thymic APC turn over physiologically and are rapidly replaced (within 2-5 wk) after lethal irradiation by donor bone marrow-derived cells. The frequency of thymocyte-DC interactions in vivo strictly correlates with thymic T cell differentiation, and is independent of the immune status of the animal. Fetal thymic APC seem to be secluded from antigen in the maternal circulation. Thymic DC-ROS probably represent the microenvironment where maturing T cells first encounter non-MHC antigens in the context of self-MHC antigens.
A sequential mouse cell culture system is described for the induction and assay of T-helper cells. Unprimed, cortisone-resistant, nylon wool- purified thymocytes cultured with adherent peritoneal exudate cells can be primed in vitro with soluble carrier protein to generate carrier- reactive helper cells. These cultured cells enhance the anti-hapten plaque-forming response of hapten-primed spleen cell cultures to hapten carrier conjugates. The culture conditions, cellular manipulations, and antigen requirements for the optimal induction of helper cells with these purified cell populations is presented. The active helper cell generated in this culture system is a thymus-derived cell which requires macrophages for its induction and must be proliferate in vitro before the manifestation of helper-cell function. Helper cells generated in vitro stimulate both carrier-specific and nonspecific enhancement of splenic anti-hapten responses. The carrier-specific and nonspecific enhancement can be distinguished by the requirement for antigen in the helper cell and spleen cell cultures, the dose of helper cells added to the spleen cell cultures, and by the requirement for additional splenic adherent accessory cell interactions.
The identification of tumor antigens recognized by cytotoxic and T helper lymphocytes has led to the development of specific cancer vaccines. Immunization with tumor antigen-pulsed dendritic cells has proved effective at eliciting elevated levels of tumor antigen – specific T cells in patient blood, but objective clinical responses remain rare, suggesting that vaccine-induced T cells are not trafficking optimally to site(s) of tumor burden. Accumulating evidence from animal models suggests that route of immunization can have a substantial influence on the subsequent migration of primed, activated T cells in vivo.
In a clinical trial designed to elicit more effective cytotoxic T-cell mediated antitumor responses, metastatic melanoma patients were immunized directly via a peripheral intralymphatic route with autologous dendritic cells pulsed with HLA-A*0201-restricted melanoma-associated peptide antigens derived from MART-1 and gp100.
Within 10 days of intralymphatic dendritic cell vaccination, four of six patients developed dramatic and diffuse erythematous rashes in sun-exposed areas of skin that showed extensive T-cell infiltration. CTLs grown from rash biopsies were strongly enriched for tumor antigen – specific T cells that had elevated expression of cutaneous lymphocyte antigen and chemokine receptor-6, consistent with a skin-homing phenotype. Of note, the only patient in the study with cutaneously localized disease showed a significant regression of metastatic lesions following the development of a surrounding rash.
The evidence presented here is consistent with immunization studies in animal models and supports the concept that T cells are “imprinted” in peripheral lymph node sites to express specific ligands and chemokine receptors that allow them to migrate to skin. Furthermore, the preferential migration of the T cells to sun-exposed cutaneous sites suggests that inflammation plays a critical role in this migration. These observations suggest that further study of the effects of immunization route and inflammation on T-cell migration in humans is warranted, and could lead to vaccination approaches that would more reliably direct trafficking of activated T cells to diverse sites of metastatic disease.
The dose of foreign antigen can influence whether a cell-mediated or humoral class of immune response is elicited, and this may be largely accounted for by the development of CD4+ T helper cells (Th) producing distinct sets of cytokines. The ability of antigen dose to direct the development of a Th1 or Th2 phenotype from naive CD4+ T cells, however, has not been demonstrated. In this report, we show that the antigen dose used in primary cultures could directly affect Th phenotype development from naive DO11.10 TCR-alpha beta-transgenic CD4+ T cells when dendritic cells or activated B cells were used as the antigen- presenting cells. Consistent with our previous findings, midrange peptide doses (0.3-0.6 microM) directed the development of Th0/Th1-like cells, which produced moderate amounts of interferon gamma (IFN-gamma). As the peptide dose was increased, development of Th1-like cells producing increased amounts of IFN-gamma was initially observed. At very high (> 10 microM) and very low (< 0.05 microM) doses of antigenic peptide, however, a dramatic switch to development of Th2-like cells that produced increasing amounts of interleukin 4 (IL-4) and diminishing levels of IFN-gamma was observed. This was true even when highly purified naive, high buoyant density CD4+ LECAM-1hi T cells were used, ruling out a possible contribution from contaminating "memory" phenotype CD4+ T cells. Neutralizing anti-IL-4 antibodies completely inhibited the development of this Th2-like phenotype at both high and low antigen doses, demonstrating a requirement for endogenous IL-4. Our findings suggest that the antigen dose may affect the levels of endogenous cytokines such as IL-4 in primary cultures, resulting in the development of distinct Th cell phenotypes.