Human Dendritic Cells Skew Isotype Switching of CD40-activated Naive B Cells towards IgA1 and IgA2

Within T cell–rich areas of secondary lymphoid organs, interdigitating dendritic cells recruit antigen-specific T cells that then induce B cells to secrete Igs. This study investigates the possible role(s) of dendritic cells in the regulation of human B cell responses. In the absence of exogenous cytokines, in vitro generated dendritic cells (referred to as Dendritic Langerhans cells, D-Lc) induced surface IgA expression on ~10% of CD40-activated naive sIgD⁺ B cells. In the presence of IL-10 and TGF-β, a combination of cytokines previously identified for its capacity to induce IgA switch, D-Lc strongly potentiated the induction of sIgA on CD40-activated naive B cells from 5% to 40–50%. D-Lc alone did not induce the secretion of IgA by CD40-activated naive B cells, which required further addition of IL-10. Furthermore, D-Lc skewed towards the IgA isotype at the expense of IgG, the Ig production of CD40-activated naive B cells cultured in the presence of IL-10 and TGF-β. Importantly, under these culture conditions, both IgA₁ and IgA₂ were detected. In the presence of IL-10, secretion of IgA2 by CD40-activated naive B cells could be detected only in response to D-Lc and was further enhanced by TGF-β. Collectively, these results suggest that in addition to activating T cells in the extrafollicular areas of secondary lymphoid organs, human D-Lc also directly modulate T cell–dependent B cell growth and differentiation, by inducing the IgA isotype switch.     

Paracrine Regulation of Germinal Center B Cell Adhesion through the c-Met–Hepatocyte Growth Factor/Scatter Factor Pathway

T cell–dependent humoral immune responses are initiated by the activation of naive B cells in the T cell areas of the secondary lymphoid tissues. This primary B cell activation leads to migration of germinal center (GC) cell precursors into B cell follicles where they engage follicular dendritic cells (FDC) and T cells, and differentiate into memory B cells or plasma cells. Both B cell migration and interaction with FDC critically depend on integrin-mediated adhesion. To date, the physiological regulators of this adhesion were unkown. In the present report, we have identified the c-met–encoded receptor tyrosine kinase and its ligand, the growth and motility factor hepatocyte growth factor/scatter factor (HGF/SF), as a novel paracrine signaling pathway regulating B cell adhesion. We observed that c-Met is predominantly expressed on CD38⁺CD77⁺ tonsillar B cells localized in the dark zone of the GC (centroblasts). On tonsil B cells, ligation of CD40 by CD40-ligand, induces a transient strong upregulation of expression of the c-Met tyrosine kinase. Stimulation of c-Met with HGF/SF leads to receptor phosphorylation and, in addition, to enhanced integrin-mediated adhesion of B cells to both VCAM-1 and fibronectin. Importantly, the c-Met ligand HGF/SF is produced at high levels by tonsillar stromal cells thus providing signals for the regulation of adhesion and migration within the lymphoid microenvironment.     

Epstein-Barr Virus–induced Molecule 1 Ligand Chemokine Is Expressed by Dendritic Cells in Lymphoid Tissues and Strongly Attracts Naive T Cells and Activated B Cells

Movement of T and B lymphocytes through secondary lymphoid tissues is likely to involve multiple cues that help the cells navigate to appropriate compartments. Epstein-Barr virus– induced molecule 1 (EBI-1) ligand chemokine (ELC/MIP3β) is expressed constitutively within lymphoid tissues and may act as such a guidance cue. Here, we have isolated mouse ELC and characterized its expression pattern and chemotactic properties. ELC is expressed constitutively in dendritic cells within the T cell zone of secondary lymphoid tissues. Recombinant ELC was strongly chemotactic for naive (L-selectin^hi) CD4 T cells and for CD8 T cells and weakly attractive for resting B cells and memory (L-selectin^lo) CD4 T cells. After activation through the B cell receptor, the chemotactic response of B cells was enhanced. Like its human counterpart, murine ELC stimulated cells transfected with EBI-1/CC chemokine receptor 7 (CCR7). Our findings suggest a central role for ELC in promoting encounters between recirculating T cells and dendritic cells and in the migration of activated B cells into the T zone of secondary lymphoid tissues.     

In Vivo Detection of Dendritic Cell Antigen Presentation to CD4+ T Cells

Although lymphoid dendritic cells (DC) are thought to play an essential role in T cell activation, the initial physical interaction between antigen-bearing DC and antigen-specific T cells has never been directly observed in vivo under conditions where the specificity of the responding T cells for the relevant antigen could be unambiguously assessed. We used confocal microscopy to track the in vivo location of fluorescent dye-labeled DC and naive TCR transgenic CD4⁺ T cells specific for an OVA peptide–I-A^d complex after adoptive transfer into syngeneic recipients. DC that were not exposed to the OVA peptide, homed to the paracortical regions of the lymph nodes but did not interact with the OVA peptide-specific T cells. In contrast, the OVA peptide-specific T cells formed large clusters around paracortical DC that were pulsed in vitro with the OVA peptide before injection. Interactions were also observed between paracortical DC of the recipient and OVA peptide-specific T cells after administration of intact OVA. Injection of OVA peptide-pulsed DC caused the specific T cells to produce IL-2 in vivo, proliferate, and differentiate into effector cells capable of causing a delayed-type hypersensitivity reaction. Surprisingly, by 48 h after injection, OVA peptide-pulsed, but not unpulsed DC disappeared from the lymph nodes of mice that contained the transferred TCR transgenic population. These results demonstrate that antigen-bearing DC directly interact with naive antigen-specific T cells within the T cell–rich regions of lymph nodes. This interaction results in T cell activation and disappearance of the DC.     

B Lymphocyte Chemotaxis Regulated in Association with Microanatomic Localization, Differentiation State, and B Cell Receptor Engagement

Migration of mature B lymphocytes within secondary lymphoid organs and recirculation between these sites are thought to allow B cells to obtain T cell help, to undergo somatic hypermutation, to differentiate into effector cells, and to home to sites of antibody production. The mechanisms that direct migration of B lymphocytes are unknown, but there is evidence that G protein–coupled receptors, and possibly chemokine receptors, may be involved. Stromal cell– derived factor (SDF)-1α is a CXC chemokine previously characterized as an efficacious chemoattractant for T lymphocytes and monocytes in peripheral blood. Here we show with purified tonsillar B cells that SDF-1α also attracts naive and memory, but not germinal center (GC) B lymphocytes. Furthermore, GC B cells could be converted to respond to SDF-1α by in vitro differentiation into memory B lymphocytes. Conversely, the migratory response in naive and memory B cells was significantly reduced after B cell receptor engagement and CD40 signaling. The receptor for SDF-1, CXC chemokine receptor 4 (CXCR4), was found to be expressed on responsive as well as unresponsive B cell subsets, but was more rapidly downregulated on responsive cells by ligand. Finally, messenger RNA for SDF-1 was detected by in situ hybridization in a layer of cells surrounding the GC. These findings show that responsiveness to the chemoattractant SDF-1α is regulated during B lymphocyte activation, and correlates with positioning of B lymphocytes within a secondary lymphoid organ.     

Antigen-dependent and -independent Ca2+ Responses Triggered in T Cells by Dendritic Cells Compared with B Cells

Dendritic cells (DCs) are much more potent antigen (Ag)-presenting cells than resting B cells for the activation of naive T cells. The mechanisms underlying this difference have been analyzed under conditions where ex vivo DCs or B cells presented known numbers of specific Ag–major histocompatibility complex (MHC) complexes to naive CD4⁺ T cells from T cell antigen receptor (TCR) transgenic mice. Several hundred Ag–MHC complexes presented by B cells were necessary to elicit the formation of a few T–B conjugates with small contact zones, and the resulting individual T cell Ca²⁺ responses were all-or-none. In contrast, Ag-specific T cell Ca²⁺ responses can be triggered by DCs bearing an average of 30 Ag–MHC complexes per cell. Formation of T–DC conjugates is Ag-independent, but in the presence of the Ag, the surface of the contact zone increases and so does the amplitude of the T cell Ca²⁺ responses. These results suggest that Ag is better recognized by T cells on DCs essentially because T–DC adhesion precedes Ag recognition, whereas T–B adhesion requires Ag recognition. Surprisingly, we also recorded small Ca²⁺ responses in T cells interacting with unpulsed DCs. Using DCs purified from MHC class II knockout mice, we provide evidence that this signal is mostly due to MHC–TCR interactions. Such an Ag-independent, MHC-triggered calcium response could be a survival signal that DCs but not B cells are able to deliver to naive T cells.     

Measles Virus Infects Human Dendritic Cells and Blocks Their Allostimulatory Properties for CD4+ T Cells

Measles causes a profound immune suppression which is responsible for the high morbidity and mortality induced by secondary infections. Dendritic cells (DC) are professional antigen-presenting cells required for initiation of primary immune responses. To determine whether infection of DC by measles virus (MV) may play a role in virus-induced suppression of cell-mediated immunity, we examined the ability of CD1a⁺ DC derived from cord blood CD34⁺ progenitors and Langerhans cells isolated from human epidermis to support MV replication. Here we show that both cultured CD1a⁺ DC and epidermal Langerhans cells can be infected in vitro by both vaccine and wild type strains of MV. DC infection with MV resulted within 24–48 h in cell–cell fusion, cell surface expression of hemagglutinin, and virus budding associated with production of infectious virus. MV infection of DC completely abrogated the ability of the cells to stimulate the proliferation of naive allogeneic CD4⁺ T cell as early as day 2 of mixed leukocyte reaction (MLR) (i.e., on day 4 of DC infection). Mannose receptor–mediated endocytosis and viability studies indicated that the loss of DC stimulatory function could not be attributed to the death or apoptosis of DC. This total loss of DC stimulatory function required viral replication in the DC since ultraviolet (UV)-inactivated MV or UV-treated supernatant from MV-infected DC did not alter the allostimulatory capacity of DC. As few as 10 MV- infected DC could block the stimulatory function of 10⁴ uninfected DC. More importantly, MV-infected DC, in which production of infectious virus was blocked by UV treatment or paraformaldehyde fixation, actively suppressed allogeneic MLR upon transfer to uninfected DC–T-cultures. Thus, the mechanisms which contribute to the loss of the allostimulatory function of DC include both virus release and active suppression mediated by MV-infected DC, independent of virus production. These data suggest that carriage of MV by DC may facilitate virus spreading to secondary lymphoid organs and that MV replication in DC may play a central role in the general immune suppression observed during measles.     

B Cells Directly Tolerize CD8+ T Cells

This report investigates the response of CD8⁺ T cells to antigens presented by B cells. When C57BL/6 mice were injected with syngeneic B cells coated with the K^b-restricted ovalbumin (OVA) determinant OVA_257–264, OVA-specific cytotoxic T lymphocyte (CTL) tolerance was observed. To investigate the mechanism of tolerance induction, in vitro–activated CD8⁺ T cells from the K^b-restricted, OVA-specific T cell receptor transgenic line OT-I (OT-I cells) were cultured for 15 h with antigen-bearing B cells, and their survival was determined. Antigen recognition led to the killing of the B cells and, surprisingly, to the death of a large proportion of the OT-I CTLs. T cell death involved Fas (CD95), since OT-I cells deficient in CD95 molecules showed preferential survival after recognition of antigen on B cells. To investigate the tolerance mechanism in vivo, naive OT-I T cells were adoptively transferred into normal mice, and these mice were coinjected with antigen-bearing B cells. In this case, OT-I cells proliferated transiently and were then lost from the secondary lymphoid compartment. These data provide the first demonstration that B cells can directly tolerize CD8⁺ T cells, and suggest that this occurs via CD95-mediated, activation-induced deletion.     

Resting Memory CD8+ T Cells are Hyperreactive to Antigenic Challenge In Vitro

The characteristics of CD8⁺ T cells responsible for memory responses are still largely unknown. Particularly, it has not been determined whether different activation thresholds distinguish naive from memory CD8⁺ T cell populations. In most experimental systems, heterogeneous populations of primed CD8⁺ T cells can be identified in vivo after immunization. These cells differ in terms of cell cycle status, surface phenotype, and/or effector function. This heterogeneity has made it difficult to assess the activation threshold and the relative role of these subpopulations in memory responses. In this study we have used F5 T cell receptor transgenic mice to generate a homogeneous population of primed CD8⁺ T cells. In the F5 transgenic mice, peptide injection in vivo leads to activation of most peripheral CD8⁺ T cells. In vivo BrdU labeling has been used to follow primed T cells over time periods spanning several weeks after peptide immunization. Our results show that the majority of primed CD8⁺ T cells generated in this system are not cycling and express increased levels of CD44 and Ly6C. These cells remain responsive to secondary peptide challenge in vivo as evidenced by short term upregulation of activation markers such as CD69 and CD44. The activation thresholds of naive and primed CD8⁺ T cells were compared in vitro. We found that CD8⁺ T cells from primed mice are activated by peptide concentrations 10–50-fold lower than naive mice. In addition, the kinetics of interleukin 2Rα chain upregulation by primed CD8⁺ T cells differ from naive CD8⁺ T cells. These primed hyperresponsive CD8⁺ T cells might play an important role in the memory response.     

Selective Recruitment of Immature and Mature Dendritic Cells by Distinct Chemokines Expressed in Different Anatomic Sites

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.     

9-O-Acetylation of Sialomucins: A Novel Marker of Murine CD4 T Cells that Is Regulated during Maturation and Activation

Terminal sialic acids on cell surface glycoconjugates can carry 9-O-acetyl esters. For technical reasons, it has previously been difficult to determine their precise distribution on different cell types. Using a recombinant soluble form of the Influenza C virus hemagglutinin-esterase as a probe for 9-O-acetylated sialic acids, we demonstrate here their preferential expression on the CD4 T cell lineage in normal B10.A mouse lymphoid organs. Of total thymocytes, 8–10% carry 9-O-acetylation; the great majority of these are the more mature PNA⁻, HSA⁻, and TCR^hi medullary cells. While low levels of 9-O-acetylation are seen on some CD4/CD8 double positive (DP) and CD8 single positive (SP) cells, high levels are present primarily on 80– 85% of CD4 SP cells. Correlation with CD4 and CD8 levels suggests that 9-O-acetylation appears as an early differentiation marker as cells mature from the DP to the CD4 SP phenotype. This high degree of 9-O-acetylation is also present on 90–95% of peripheral spleen and lymph node CD4 T cells. In contrast, only a small minority of CD8 T cells and B cells show such levels of 9-O-acetylation. Among mature peripheral CD4 T lymphocytes, the highly O-acetylated cells are Mel 14^hi, CD44^lo, and CD45R(exon B)^hi, features typical of naive cells. Digestions with trypsin and O-sialoglycoprotease (OSGPase) and ELISA studies of lipid extracts indicate that the 9-O-acetylated sialic acids on peripheral CD4 T cells are predominantly on O-linked mucintype glycoproteins and to a lesser degree, on sialylated glycolipids (gangliosides). In contrast, sialic acids on mucin type molecules of CD8 T cells are not O-acetylated; instead these molecules mask the recognition of O-acetylated gangliosides that seem to be present at similar levels as on CD4 cells. The 9-O-acetylated gangliosides on mouse T cells are not bound by CD60 antibodies, which recognize O-acetylated gangliosides in human T cells. Tethering 9-O-acetylated mucins with the Influenza C probe with or without secondary cross-linking did not cause activation of CD4 T cells. However, activation by other stimuli including TCR ligation is associated with a substantial decrease in surface 9-O-acetylation, primarily in the mucin glycoprotein component. Thus, 9-O-acetylation of sialic acids on cell surface mucins is a novel marker on CD4 T cells that appears on maturation and is modulated downwards upon activation.     

CD40 Ligation on Human Cord Blood CD34+Hematopoietic Progenitors Induces Their Proliferation and Differentiation into Functional Dendritic Cells

Human CD34⁺ multilineage progenitor cells (CD34HPC) from cord blood and bone marrow express CD40, a member of the tumor necrosis factor–receptor family present on various hematopoietic and nonhematopoietic cells. As hyper-IgM patients with mutated CD40 ligand (CD40L) exhibit neutropenia, no B cell memory, and altered T cell functions leading to severe infections, we investigated the potential role of CD40 on CD34HPC development. CD40activated cord blood CD34HPC were found to proliferate and differentiate independently of granulocyte/macrophage colony-stimulating factor, into a cell population with prominent dendritic cell (DC) attributes including priming of allogeneic naive T cells. DC generated via the CD40 pathway displayed strong major histocompatibility complex class II DR but lacked detectable CD1a and CD40 expression. These features were shared by a dendritic population identified in situ in tonsillar T cell areas. Taken together, the present data demonstrate that CD40 is functional on CD34HPC and its cross-linking by CD40L⁺ cells results in the generation of DC that may prime immune reactions during antigen-driven responses to pathogenic invasion, thus providing a link between hematopoiesis, innate, and adaptive immunity.The CD34⁺ compartment of human progenitor cells displays multilineage hematopoietic capacities, including the generation of dendritic cells (DC) (1, 2). Human CD34⁺ multilineage progenitor cells (CD34HPC)¹ express CD40 (3), a member of the TNFR/NGFR family instrumental in the activation, growth, and survival of various hematopoietic and nonhematopoietic cells (4–7). CD40 ligation induces proliferation and survival of B lymphocytes, turns on their isotype-switch machinery, and directs the generation of memory B cells (8–12). CD40 triggering also activates monocytes (13), mature DCs (14), endothelial cells (15, 16), fibroblasts (17), and induces cytokine production by thymic endothelial cells (18). However, in addition to mature B cells, only progenitor B cells (19) and fibroblasts have been shown to proliferate under CD40 stimulation.The importance of CD40–CD40 ligand (CD40L) interactions in vivo has been revealed in hyper-IgM (HIM) patients bearing a mutated CD40L on T cells (20–24). As a consequence, patients suffer from various bacterial and viral infections as well as from Cryptosporidial diarrhea and Pneumocystis carinii pneumonia. These infections may be the consequence of several primary immunodeficiencies, including defective B cell memory (excess of IgM and low or absent secondary Ab responses), altered T cell responses, and neutropenia (25). Because of the prominent link of CD40 with cell proliferation and differentiation, and because of the defective humoral, cell-mediated immunity and granulopoietic responses observed in HIM patients, we engaged into the analysis of CD40 function on CD34HPC. The data show that CD40 ligation induces CD34HPC to proliferate and differentiate into cells with dendritic cell attributes.     

Memory B Cells Are Biased Towards Terminal Differentiation: A Strategy That May Prevent Repertoire Freezing

Isolation of large numbers of surface IgD⁺CD38⁻ naive and surface IgD⁻CD38⁻ memory B cells allowed us to study the intrinsic differences between these two populations. Upon in vitro culture with IL-2 and IL-10, human CD40–activated memory B cells undergo terminal differentiation into plasma cells more readily than do naive B cells, as they give rise to five- to eightfold more plasma cells and three- to fourfold more secreted immunoglobulins. By contrast, naive B cells give rise to a larger number of nondifferentiated B blasts. Saturating concentrations of CD40 ligand, which fully inhibit naive B cell differentiation, only partially affect that of memory B cells. The propensity of memory B cells to undergo terminal plasma cell differentiation may explain the extensive extra follicular plasma cell reaction and the limited germinal center reaction observed in vivo after secondary immunizations, which contrast with primary responses in carrier-primed animals. This unique feature of memory B cells may confer two important capacities to the immune system: (a) the rapid generation of a large number of effector cells to efficiently eliminate the pathogens; and (b) the prevention of the overexpansion and chronic accumulation of one particular memory B cell clone that would freeze the available peripheral repertoire.     

Feasibility of CTLA4Ig gene delivery and expression in vivo using retrovirally transduced myeloid dendritic cells that induce alloantigen-specific T cell anergy in vitro

Dendritic cells (DC) are highly specialised, bone marrow (BM)-derived antigen-presenting cells (APC) that initiate and regulate immune responses. They provide costimulatory signals (in particular, CD40 and the CD28 ligands CD80 and CD86) necessary for naive T cell activation. Functional expression of CD80 and CD86 is blocked by the fusion protein cytotoxic T lymphocyte antigen 4-immunoglobulin (CTLA4Ig), that promotes tolerance induction in animals. Here, replicating mouse (B10; H2b) myeloid DC progenitors, were retrovirally transduced to express CTLA4Ig using the centrifugal enhancement method. Gene product was detected by immunocyto- or histochemistry. Maximal DC transduction efficiency was 62%. Compared with control, zeomycin-resistance gene (Zeo)-transduced DC, CTLA4Ig-expressing cells showed markedly impaired capacity to stimulate naive allogeneic (C3H; H2k) T cell proliferation and cytotoxic T lymphocyte (CTL) generation. Their ability to induce alloantigen-specific T cell hyporesponsiveness was reversed by exogenous IL-2 in secondary mixed leukocyte reactions (MLR). Following local (s.c.) transfer to allogeneic recipients, the genetically modified DC trafficked to T cell areas of draining lymphoid tissue, where transgene expression was detected. Ex vivo analysis of proliferative and CTL responses revealed donor-specific inhibition of alloimmune reactivity by the CTLA4Ig-transduced DC. This effect was associated with marked inhibition of interferon (IFN)-gamma production, but significant augmentation of IL-4 and IL-10 secretion. Thus, retroviral transduction of DC permits in vivo delivery of CTLA4Ig to the precise microenvironment where antigen (Ag) presentation occurs. Comparatively nonimmunogenic retroviral vectors, that allow permanent transgene expression in DC, and promote localized delivery of the immunosuppressive transgene product, promote immune deviation and Ag-specific T cell hyporesponsiveness.     

Dendritic Cell Development in Culture from Thymic Precursor Cells in the Absence of Granulocyte/Macrophage Colony-stimulating Factor

The earliest lymphoid precursor population in the adult mouse thymus had previously been shown to produce not only T cells, but also dendritic cell (DC) progeny on transfer to irradiated recipients. In this study, culture of these isolated thymic precursors with a mixture of cytokines induced them to proliferate and to differentiate to DC, but not to T lineage cells. At least 70% of the individual precursors had the capacity to form DC. The resultant DC were as effective as normal thymic DC in the functional test of T cell stimulation in mixed leukocyte cultures. The cultured DC also expressed high levels of class I and class II major histocompatibility complex, together with CD11c, DEC-205, CD80, and CD86, markers characteristic of mature DC in general. However, they did not express CD8α or BP-1, markers characteristic of normal thymic DC. The optimized mixture of five to seven cytokines required for DC development from these thymic precursors did not include granulocyte/macrophage colony stimulating factor (GM-CSF), usually required for DC development in culture. The addition of anti–GM-CSF antibody or the use of precursors from GM-CSF–deficient mice did not prevent DC development. Addition of GM-CSF was without effect on DC yield when interleukin (IL) 3 and IL-7 were present, although some stimulation by GM-CSF was noted in their absence. In contrast, DC development was enhanced by addition of the Flt3/Flk2 ligand, in line with the effects of the administration of this cytokine in vivo. The results indicate that the development of a particular lineage of DC, probably those of lymphoid precursor origin, may be independent of the myeloid hormone GM-CSF.Dendritic cells (DC)¹ (1) are generally considered as relatives of monocytes and macrophages and to be of myeloid origin. Strong support for this view comes from many studies on the outgrowth of DC in culture, induced principally by GM-CSF, usually in association with other cytokines including TNF-α (2–11; for review see reference 12). The DC appear to derive from a progenitor also capable of forming granulocytes and macrophages (13), although more recently a committed DC progenitor, possibly a downstream precursor, has been identified (14). The myeloid nature of DC is emphasized by the direct development of a form of DC from blood monocytes (9, 15–17; for review see reference 12). All these lines of evidence for a myeloid origin of DC derive from culture studies using GM-CSF.In contrast to these studies, we have used adoptive transfer of highly purified precursor cells isolated from the mouse thymus to demonstrate that certain types of DC are related to the lymphoid lineage. The earliest T precursor population isolated from the adult mouse thymus, the “low CD4 precursor,” was unable to form detectable erythroid or myeloid cells, yet had the potential to form T cells, B cells, NK cells, and DC (18–23; for review see reference 24). A progenitor cell with similar developmental potential has since been isolated from human bone marrow (25). T cells and DC bearing CD8α, a characteristic of murine thymic DC (26), developed in parallel when this low CD4 precursor was transferred directly into a recipient thymus (21). We have recently found that a downstream thymic precursor (CD4⁻8⁻44⁺25⁺c-kit⁺), now no longer able to form B cells or NK cells, still retains full capacity to form DC as well as T cells, suggesting a strong relationship between the two lineages (27).These thymic precursors also formed CD8α⁺ DC in the spleen after intravenous transfer, suggesting that the CD8α⁺ DC normally found in peripheral lymphoid tissues might also be of lymphoid origin. These CD8⁺ splenic DC appear to have a regulatory role in T cell responses (28).Our initial attempts to grow the thymic low CD4 precursors in culture under the influence of multiple combinations of up to three cytokines were unsuccessful. Some growth and development was obtained using an underlay of a thymic epithelial cell line; under these conditions, a limited production of DC was evident (29). However, more extensive growth, with development into DC rather than T cell progeny, was evident once a more complex cocktail of cytokines was used. It was notable that GMCSF was not required for this DC development in culture.     

Germinal Center Founder Cells Display Propensity for Apoptosis before Onset of Somatic Mutation

B lymphocytes undergo affinity maturation of their antigen receptors within germinal centers. These anatomical structures develop in secondary lymphoid organs from the clonal expansion of a few antigen-specific founder B cells, whose isolation and characterization are reported here. Human germinal center founder cells express the naive B cell markers surface IgM and IgD as well as the germinal center B cell markers CD10 and CD38. They express low levels of Bcl-2, high levels of Fas, and undergo rapid apoptosis in culture. The smaller nonproliferating sIgM⁺IgD⁺CD38⁺ B cells displayed a lower level of somatic mutation in their immunoglobulin variable region genes compared with the large proliferating ones. Unmutated sIgM⁺IgD⁺CD38⁺ tonsillar B cells may thus represent germinal center founder cells in which the program for apoptotic cell death is triggered before the onset of somatic mutation, allowing the selection of the germline antibody repertoire at an early stage.     

In Vivo Microbial Stimulation Induces Rapid CD40 Ligand–independent Production of Interleukin 12 by Dendritic Cells and their Redistribution to T Cell Areas

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.Interleukin 12 (IL-12) is a key cytokine in the induction of cell-mediated immunity to intracellular pathogens. In the innate response to these microbial agents, IL-12 triggers the production of IFN-γ and TNF from unsensitized NK and T cells. At the same time, IL-12 selectively promotes the differentiation of Th1 CD4⁺ cells, which produce the same effector lymphokines upon restimulation with antigen. Thus, the induction of IL-12 early in infection initiates innate resistance to the pathogen while ensuring the induction of the correct class of adaptive host response (1).Macrophages (MΦ)¹ activated by microbial stimulation produce high levels of IL-12, and it has been assumed that these cells provide the major source of the cytokine in Th1 response initiation (2). Indeed, in vitro studies with TCR transgenic CD4⁺ cells primed by antigen-pulsed cells showed IL-12–producing MΦ to be highly effective in inducing selective Th1 cell differentiation (3). However, the model of class selection suggested by these experiments requires that both antigen-bearing dendritic cells (DC) and IL-12–producing MΦ travel from the site of infection to lymphoid tissues where the responding T cells are found, and at present, no evidence exists for such MΦ migration. Furthermore, a model in which IL-12 produced by MΦ acts in a paracrine fashion to drive Th1 development of microbe-specific T cells would lead to Th1 differentiation of all recently activated precursors in the local microenvironment, thus limiting the ability of the immune system to independently control Th1 responses to different antigens and potentially leading to inflammatory responses to self-antigens. A similar objection applies to models of type 1 response initiation involving other “third-party” IL-12–producing cells such as neutrophils (4).In contrast to MΦ, DC constitute a highly efficient system for capturing antigens in the periphery and delivering them to the T cell areas of lymphoid tissues (5, 6). It is believed that this allows perusal of peripheral antigens by T cells that recirculate between the blood and lymphoid compartments. In addition, DC possess many specializations that allow them to function as efficient APCs, such as high levels of MHC products, adhesion and costimulatory molecules, extensive surface area, and high motility (5, 6). These properties suggest that DC act as the priming APC for most T cell responses and thus would be ideally placed to produce IL-12 at a site where it acts directly on those T cells responding to DC-presented immunogenic MHC–peptide complexes derived from infectious agents.The early synthesis of IL-12 is of crucial importance in determining both innate and adaptive host resistance to the intracellular protozoan, Toxoplasma gondii, and live replicative forms (tachyzoites) as well as parasite extracts have been shown to be potent inducers of the cytokine from peritoneal inflammatory MΦ in vitro (7–10). Nevertheless, inflammatory MΦ populations that are elicited by local injection of an irritant, or, for that matter, any population obtained after in vivo or in vitro manipulation, may not be representative of the naive cells that initiate IL-12 responses. Therefore, we have used immunolocalization techniques to identify the cells first making IL-12 after in vivo stimulation with T. gondii products or LPS. Our results clearly demonstrate that DC, not MΦ, are the cells involved in the IL-12 response to these microbial stimuli, which also simultaneously trigger DC recruitment to the T cell areas of the spleen. Together, these findings support a two cell model of in vivo T cell activation in which the DC serves as both the APC and the IL-12–producing initiator of the Th1 response.     

On the Key Role of Secondary Lymphoid Organs in Antiviral Immune Responses Studied in Alymphoplastic (aly/aly) and Spleenless (Hox11−/−) Mutant Mice

The role of the spleen and of other organized secondary lymphoid organs for the induction of protective antiviral immune responses was evaluated in orphan homeobox gene 11 knockout mice (Hox11^−/−) lacking the spleen, and in homozygous alymphoplastic mutant mice (aly/aly) possessing a structurally altered spleen but lacking lymph nodes and Peyer's patches. Absence of the spleen had no major effects on the immune response, other than delaying the antibody response by 1–2 d. In aly/aly mice, the thymus-independent IgM response against vesicular stomatitis virus (VSV) was delayed and reduced, whereas the T-dependent switch to the protective IgG was absent. Therefore, aly/aly mice were highly susceptible to VSV infection. Since aly/aly spleen cells yielded neutralizing IgM and IgG after adoptive transfer into recipients with normally structured secondary lymphoid organs, these data suggest that the structural defect was mainly responsible for inefficient T–B cooperation. Although aly/aly mice generated detectable, but reduced, CTL responses after infection with vaccinia virus (VV) and lymphocytic choriomeningitis virus (LCMV), the elimination of these viruses was either delayed (VV) or virtually impossible (LCMV); irrespective of the dose or the route of infection, aly/aly mice developed life-long LCMV persistence. These results document the critical role of organized secondary lymphoid organs in the induction of naive T and B cells. These structures also provide the basis for cooperative interactions between antigen-presenting cells, T cells, and B cells, which are a prerequisite for recovery from primary virus infections via skin or via blood.     

Predominant Role for Directly Transfected Dendritic Cells in Antigen Presentation to CD8+ T Cells after Gene Gun Immunization

Cutaneous gene (DNA) bombardment results in substantial expression of the encoded antigen in the epidermal layer as well as detectable expression in dendritic cells (DC) in draining lymph nodes (LNs). Under these conditions, two possible modes of DC antigen presentation to naive CD8⁺ T cells might exist: (a) presentation directly by gene-transfected DC trafficking to local lymph nodes, and (b) cross-presentation by untransfected DC of antigen released from or associated with transfected epidermal cells. The relative contributions of these distinct modes of antigen presentation to priming for cytotoxic T cell (CTL) responses have not been clearly established. Here we show that LN cells directly expressing the DNA-encoded antigen are rare; 24 h after five abdominal skin bombardments, the number of these cells does not exceed 50–100 cells in an individual draining LN. However, over this same time period, the total number of CD11c⁺ DC increases more than twofold, by an average of 20,000–30,000 DC per major draining node. This augmentation is due to gold bombardment and is independent of the presence of plasmid DNA. Most antigen-bearing cells in the LNs draining the site of DNA delivery appear to be DC and can be depleted by antibodies to an intact surface protein encoded by cotransfected DNA. This finding of predominant antigen presentation by directly transfected cells is also consistent with data from studies on cotransfection with antigen and CD86-encoding DNA, showing that priming of anti-mutant influenza nucleoprotein CTLs with a single immunization is dependent upon coexpression of the DNAs encoding nucleoprotein and B7.2 in the same cells. These observations provide insight into the relative roles of direct gene expression and cross-presentation in CD8⁺ T cell priming using gene gun immunization, and indicate that augmentation of direct DC gene expression may enhance such priming.