Development of lymphocytes in interleukin 7-transgenic mice

We have developed and established mouse transgenic lines in which the mouse interleukin 7 gene was targeted for expression in the lymphoid cell compartment. Northern blot analysis indicate that the transgene is expressed in bone marrow (BM), spleen and thymus, but not in kidney, liver, brain or heart. Both the frequency and absolute numbers of B cell precursors and mature B lymphocytes are increased in the BM and spleen of the transgenic mice. Although there is no expansion of the pro-T lymphocyte population in the BM, the number of all major subsets of thymocytes and peripheral T lymphocytes is increased in the majority of the transgenic mice analyzed. The B and T cell lymphocytes in the transgenic mice are functionally competent. In contrast, the number of granulocytes and macrophages in the BM of transgenic mice is similar to that in control non-transgenic littermates. Our results indicate that interleukin 7 plays an important role in vivo in the development of B and T lymphocytes.     

A homodimeric complex of HLA-G on normal trophoblast cells modulates antigen-presenting cells via LILRB1

In healthy individuals, the non-classical MHC molecule HLA-G is only expressed on fetal trophoblast cells that invade the decidua during placentation. We show that a significant proportion of HLA-G at the surface of normal human trophoblast cells is present as a disulphide-linked homodimer of the conventional beta(2)m-associated HLA-I complex. HLA-G is a ligand for leukocyte immunoglobulin-like receptors (LILR), which bind much more efficiently to dimeric HLA-G than to conventional HLA-I molecules. We find that a LILRB1-Fc fusion protein preferentially binds the dimeric form of HLA-G on trophoblast cells. We detect LILRB1 expression on decidual myelomonocytic cells; therefore, trophoblast HLA-G may modulate the function of these cells. Co-culture with HLA-G(+) cells does not inhibit monocyte-derived dendritic cell up-regulation of HLA-DR and costimulatory molecules on maturation, but did increase production of IL-6 and IL-10. Furthermore, proliferation of allogeneic lymphocytes was inhibited by HLA-G binding to LILRB1/2 on responding antigen-presenting cells (APC). As HLA-G is the only HLA-I molecule that forms beta(2)m-associated dimers with increased avidity for LILRB1, this interaction could represent a placental-specific signal to decidual APC. We suggest that the placenta is modulating maternal immune responses locally in the uterus through HLA-G, a trophoblast-specific, monomorphic signal present in almost every pregnancy. See accompanying commentary: (http://dx.doi.org/10.1002/eji.200737515).     

Mouse bone marrow contains large numbers of functionally competent neutrophils

The mouse has become an important model for immunological studies including innate immunity. Creating transgenic mice offers unique possibilities to study gene-function relationships. However, relatively little is known about the physiology of neutrophils from wild-type mice. Do they behave like human neutrophils, or are there species-specific differences that need to be considered when extrapolating results from mice to humans? How do we isolate neutrophils from mice? For practical reasons, many studies on mouse neutrophils are done with bone marrow cells. However, human bone marrow neutrophils appear to be heterogeneous and functionally immature. We have isolated and compared neutrophils from mouse bone marrow and from peripheral blood obtained by tail bleeding. Using the same Percoll density gradient for both preparations, we have obtained morphologically mature neutrophils from bone marrow and blood. Both cell populations responded to formylmethionyl-leucyl-phenylalanine (fMLF) with primary and secondary granule release and superoxide production. Quantitative analysis of our data revealed minor differences between cells from bone marrow and blood. Superoxide production and primary granule release were stimulated at lower fMLF concentrations in blood neutrophils. However, the amplitude and the kinetics of maximal responses were similar. The principal difference was the lifespan of the two cell populations. Bone marrow cells survived significantly longer in culture, which may suggest that they are receiving antiapoptic signals that are absent in the blood. Our data suggest that mice have a large reservoir of functionally competent neutrophils in their bone marrow. This reservoir may be needed to replace circulating neutrophils rapidly during infection.     

Bruton's tyrosine kinase defect in dendritic cells from X-linked agammaglobulinaemia patients does not influence their differentiation,maturation and antigen-presenting cell function

X‐linked agammaglobulinaemia (XLA) is a primary immunodeficiency disease characterized by very low levels or even absence of circulating antibodies. The immunological defect is caused by deletions or mutations of Bruton's tyrosine kinase gene (Btk), whose product is critically involved in the maturation of pre‐B lymphocytes into mature B cells. Btk is expressed not only in B lymphocytes but also in cells of the myeloid lineage, including dendritic cells (DC). These cells are professional antigen presenting cells (APC) that play a fundamental role in the induction and regulation of T‐cell responses. In this study, we analysed differentiation, maturation, and antigen‐presenting function of DC derived from XLA patients (XLA‐DC) as compared to DC from age‐matched healthy subjects (healthy‐DC). We found that XLA‐DC normally differentiate from monocyte precursors and mature in response to lipopolysaccharide (LPS) as assessed by de novo expression of CD83, up‐regulation of MHC class II, B7·1 and B7·2 molecules as well as interleukin (IL)‐12 and IL‐10 production. In addition, we demonstrated that LPS stimulated XLA‐DC acquire the ability to prime naïve T cells and to polarize them toward a Th1 phenotype, as observed in DC from healthy donors stimulated in the same conditions. In conclusion, these data indicate that Btk defect is not involved in DC differentiation and maturation, and that XLA‐DC can act as fully competent antigen presenting cells in T cell‐mediated immune responses.     

Ins and outs of dendritic cells

Dendritic cells (DC) are professional antigen-presenting cells which are strategically positioned at the boundaries between the inner and the outside world, in this way bridging innate and adaptive immunity. DC can initiate T cell responses against microbial pathogens and tumors due to their capacity to stimulate na?ve T cells. The development of DC occurs in distinct stages. DC precursors develop in the bone marrow and home to a large variety of tissues. Immature DC capture antigen (Ag) and, following proinflammatory signals, migrate to the lymphoid organs where, after maturation, they present captured Ag to na?ve T cells, thereby inducing differentiation of na?ve T cells into effector T cells. An important cognate event in the development of cell-mediated immunity is the interaction between CD40 and CD40 ligand. Ligation of CD40 on DC by its ligand results in maturation of the DC. In addition to CD40 ligand (expressed by activated Th cells), inflammatory cytokines, bacterial components or Ag-Ab immune complexes can induce maturation of DC. Maturation of DC is crucial for the priming of efficient T cell responses and is characterized by a decreased Ag processing capacity, an increased cell surface expression of MHC and costimulatory molecules, and rearrangement of cytoskeleton, adhesion molecules, and cytokine receptors. Mature DC migrate from peripheral tissues to secondary lymphoid organs, where T cell priming occurs. DC are not only critical in initiating T cell immunity, they also play a role in the induction of T cell tolerance and the regulation of the type of T cell response that is induced. Here we give an overview of the dendritic cell system.     

Limitations with in vitro production of dendritic cells using cytokines

Dendritic cells (DC) are the most effective antigen-presenting cells. Many studies now show that DC can be generated in vitro from a number of starting cell populations containing hematopoietic precursors. The protocols used involve different combinations of cytokines including granulocyte macrophage-colony stimulating factor (GM-CSF), which supports myeloid precursors, or interleukin-7, which supports lymphoid precursors. DC are commonly generated by in vitro culture of bone marrow or monocytes with GM-CSF and other cytokines. However, these cultures do not sustain DC production for long periods of time and do not allow the identification or study of intermediate stages in cell development. In vitro cytokine-dependent cultures of DC precursors do provide a reliable source of DC for stimulating immune responses. However, use of cells produced in cytokine-dependent cultures for the study of DC differentiation is limited, as DC development in vivo differs in cytokine dependency.     

Increased islet antigen presentation leads to type-1 diabetes in mice with autoimmune susceptibility

Granulocyte-macrophage colony-stimulating factor (GM-CSF) is frequently used in preclinical and clinical protocols to modulate autoimmune responses, bone marrow transplants, and recovery from immune ablative therapies. The immunological outcome of such therapies is not fully understood. We tested the hypothesis that GM-CSF would enhance the maturation of antigen-presenting cells, facilitating presentation of beta-cell autoantigens to autoreactive T cells. We found that islet expression of GM-CSF greatly enhanced disease in male mice. Islet-derived APC but not splenic APC showed markedly enhanced capacity to stimulate in vitro proliferative responses of islet-antigen-specific autoreactive T cells. In vivo transfer of CD8(+) and CD4(+) T cells demonstrate that autoreactive T cells undergo extensive division in pancreatic lymph nodes of GM-CSF-transgenic mice compared with wild-type NOD male mice. Together, the results presented here demonstrate that expression of GM-CSF in the pancreas can enhance autoimmunity in disease-susceptible mice.     

Toward homeostasis: regulatory dendritic cells from the bone marrow of mice with inflammation of the airways and peritoneal cavity

Inflammatory mediators from peripheral tissues may control dendritic cell (DC) development in the bone marrow. In this study, DCs (CD11c(+) cells) differentiated from the bone marrow of mice with inflammation of the airways, or the peritoneal cavity had poor priming ability resulting in reduced, long-lived responses to that antigen in vivo. This indicates enhancement of regulatory mechanisms of immune responses through a peripheral tissue-bone marrow axis. If CD11c(+) cells, expanded from the bone marrow of mice with tissue inflammation were antigen pre-loaded and injected into mice already sensitized to that antigen, then subsequent contact hypersensitivity responses were significantly reduced. The effects of inflammation were imprinted in vivo and were independent of in vitro culture conditions for DC differentiation. The effect of tissue inflammation on the bone marrow DC precursors was not detected in mice treated subcutaneously with slow-release indomethacin pellets, suggesting a role for prostanoids, including prostaglandin E(2), in differentiation of regulatory CD11c(+) cells from bone marrow. Our study represents an important homeostatic process with potential for therapeutic use in the future.     

Effect of a Trichinella spiralis infection on the distribution of mast cell precursors in tissues of thymus-bearing and non-thymus-bearing (nude) mice determined by an in vitro assay.

The frequency of precursor cells capable of giving rise to cells with characteristics of mucosal mast cells in tissues from thymus-bearing and non-thymus-bearing (nude) mice orally infected with Trichinella spiralis was determined with an in vitro assay. Analysis of the frequency of mast cell precursors in bone marrow, blood, spleen and small intestinal tissue revealed similar frequencies of mast cell precursors in bone marrow from both thymus-bearing and athymic mice. These frequencies in bone marrow were not affected by infection. However, in blood and spleen from thymus-bearing mice at Day 7 post-infection (p.i.), and in the gut at Day 14 p.i., significant increases of mast cell precursor frequencies were detected. In contrast, no significant increase was observed in the tissues of infected nude mice. These data are in accordance with in vivo findings, indicating that a mucosal mast cell response in the gut is both thymus and antigen dependent. It was concluded that a mucosal mast cell response to infection with T. spiralis is probably due to local proliferation and maturation of residing mast cell precursors, that this response might be amplified by an influx of precursor cells from the blood into the gut, and that both phenomena are T-cell dependent.     

The role of antigen-presenting cells and interleukin-12 in the priming of antigen-specific CD4+ T cells by immune stimulating complexes

Immune stimulating complexes (ISCOMs) containing the saponin adjuvant Quil A are vaccine adjuvants that promote a wide range of immune responses in vivo, including delayed-type hypersensitivity (DTH) and the secretion of both T helper 1 (Th1) and Th2 cytokines. However, the antigen-presenting cell (APC) responsible for the induction of these responses has not been characterized. Here we have investigated the role of dendritic cells (DC), macrophages (Mφ) and B cells in the priming of antigen-specific CD4⁺ T cells in vitro by ISCOMs containing ovalbumin (OVA). OVA ISCOMs pulsed bone marrow (BM)-derived DC but not BM Mφ, nor naïve B cells prime resting antigen-specific CD4⁺ T cells, and this response is greatly enhanced if DC are activated with lipopolysaccharide (LPS). Of the APC found in the spleen, only DC had the capacity to prime resting antigen specific CD4⁺ T cells following exposure to OVA ISCOMs in vitro, while Mφ and B cells were ineffective. DC, but not B cells purified from the draining lymph nodes of mice immunized with OVA ISCOMs also primed resting antigen-specific CD4⁺ T cells in vitro, suggesting that DC are also critical in vivo. Using DC and T cells from interleukin (IL)-12 p40^−/− mice, we also identified a crucial role for IL-12 in the priming of optimal CD4⁺ T cell responses by OVA ISCOMs. We suggest that DC are the principal APC responsible for the priming of CD4⁺ T cells by ISCOMs in vivo and that directed targeting of these vectors to DC may enhance their efficancy as vaccine adjuvants.     

LPS induces rapid IL-10 release by M-CSF-conditioned tolerogenic dendritic cell precursors

Dendritic cells (DC) obtained by culturing myeloid precursors in GM-CSF undergo maturation and induce an efficient T cell response when stimulated with microbial products. DC precursors themselves also recognize microbial products, and it remains unclear how these stimulated DC precursors modulate the immune response. We show here that M-CSF-conditioned human DC precursors responded to LPS, Mycobacteria bovis, and inflammatory cytokines by a rapid and robust production of IL-10, largely superior to that observed with immature DC or monocytes. The endogenous IL-10 restrained the DC precursors from converting into professional APC, as blocking the IL-10 receptor in the presence of LPS resulted in the formation of efficient T cell stimulators. LPS stimulation concomitant with DC differentiation gave rise to immature DC, which were tolerant to a secondary LPS exposure. Furthermore, the LPS-activated DC precursors reduced bystander DC maturation and anti-CD3/CD28-triggered T cell activation. These data suggest that when exposed to inflammatory or microbial signals, M-CSF-conditioned DC precursors can participate in the modulation of inflammation and immune response by rapid release of IL-10.     

Dendritic cell/macrophage precursors capture exogenous antigen for MHC class I presentation by dendritic cells

Presentation of MHC class I antigens by professional antigen-presenting cells (APC) is an important pathway in priming cytotoxic T lymphocyte responses in vivo. This study sought to identify the nature of the professional APC responsible for indirect class I presentation by examining a special feature of professional APC, namely their ability to process exogenous forms of antigen for class I presentation. Incubation of highly purified bone marrow-derived precursor cells with chicken ovalbumin (OVA) led to the efficient presentation of the major class I-restricted OVA determinant by mature dendritic cells (DC), but not by macrophages (Mϕ) derived from the precursor population. DC as well as macrophages were, however, able to mediate class II presentation of OVA, suggesting that macrophages were deficient in class I processing but not in capturing exogenous OVA. The majority of mature DC, i.e. over 80 %, generated from the precursor cells pulsed with OVA, presented the class I OVA epitope. Upon maturation, class I presentation of OVA by DC was greatly reduced, suggesting that class I processing of exogenous antigen is modulated during DC maturation in a manner similar to class II antigen processing. This study shows that bone marrow-derived DC/Mϕ progenitors capture exogenous antigen for class I presentation, and that cells of the DC lineage can be functionally distinguished from cells of the macrophage lineage based on their ability to process exogenous antigen for class I presentation.     

Soluble HLA-G molecules impair natural killer/dendritic cell crosstalk via inhibition of dendritic cells

HLA-G molecules are known to exert immunosuppressive action on DC maturation and on NK cells, and can in consequence inhibit respectively T cell responses and NK cytolysis. In this study, we show that monocyte-derived DC, differentiated in the presence of GM-CSF and IL-4, are sensitive to soluble (s) HLA-G molecules during LPS/IFN-gamma maturation as demonstrated by the decrease of CD80 and HLA-DR expressions and IL-12 secretion. Moreover, DC pretreated with sHLA-G were found to activate NK/DC crosstalk less than non-treated DC. Early activation of NK cells co-cultured with autologous DC was diminished as assessed by CD69 expression. The IFN-gamma production was impaired whereas a slight inhibition of the NK cell cytotoxicity against Daudi cell line was observed. Since sHLA-G is expressed in grafts or sites of tumour proliferation, its indirect action on NK cells via DC could constitute a pathway of early inhibition for both innate and specific immune responses.     

Bacterial DNA and immunostimulatory CpG oligonucleotides trigger maturation and activation of murine dendritic cells

Bacterial DNA and immunostimulatory (i.s.) synthetic CpG-oligodeoxynucleotides (ODN) act as adjuvants for Th1 responses and cytotoxic T cell responses to proteinaceous antigens. Dendritic cells (DC) can be referred to as “nature's adjuvant” since they display the unique capacity to sensitize naive T cells. Here, we demonstrate that bacterial DNA or i.s. CpG-ODN cause simultaneous maturation of immature DC and activation of mature DC to produce cytokines. These events are associated with the acquisition of professional antigen-presenting cell (APC) function. Unfractionated murine bone marrow-derived DC and FACS^®-fractionated MHC class II^low (termed immature DC) or MHC class II^high populations (termed mature DC) were stimulated with bacterial DNA or i.s. CpG-ODN. Similar to lipopolysaccharide, i.s. CpG-ODN caused up-regulation of MHC class II, CD40 and CD86, but not CD80 on immature and mature DC. In parallel both DC subsets were activated to produce large amounts of IL-12, IL-6 and TNF-α. CpG-ODN-activated DC displayed professional APC function in allogeneic mixed lymphocyte reaction and in staphylococcal enterotoxin B-driven naive T cell responses. We interpret these findings to mean that bacterial DNA and i.s. CpG-ODN cause maturation (first step) and activation (second step) of DC to bring about conversion of immature DC into professional APC.     

Development of dendritic cells from GM-CSF-/- mice in vitro : GM-CSF enhances production and survival of cells

The production of dendritic cells (DC) from haemopoietic progenitors maintained in long term stroma-dependent cultures (LTC) of spleen or bone marrow (BM) occurs independently of added granulocyte/macrophage colony stimulating factor (GM-CSF). The possibility that cultures depend on endogenous GM-CSF produced in low levels was tested by attempting to generate LTC from spleen and BM of GM-CSF-/- mice. Multiple cultures from GM-CSF-/- and wild type mice were established and compared for cell production. GM-CSF-/- LTC developed more slowly, but by 16 weeks produced cells resembling DC in numbers comparable to wild type cultures. LTC maintained distinct populations of small and large cells, the latter resembling DC. Cells collected from GM-CSF-/- LTC were capable antigen presenting cells (APC) for T cell stimulation and morphologically resembled DC. Large cells expressed the CD11b, CD11c, CD86, 33D1 and Dec-205 markers of DC. Addition of GM-CSF to GM-CSF-/- LTC increased the proportion of large, mature DC present in culture. Stromal cells from GM-CSF-/- LTC could support the differentiation of DC from early progenitors maintained in LTC without addition of GM-CSF. However, GM-CSF is not a critical factor in the in vitro generation of DC from progenitors. It can, however, substitute for stromal cells in increasing the survival of mature DC.     

Generation of purified stromal cell cultures that support lymphoid and myeloid precursors

Treatment of Dexter-type long-term bone marrow cultures with the antibiotic mycophenolic acid (MPA) eliminates all hemopoietic cells from the cultures, while a morphologically intact, adherent stromal cell layer is retained. The ability of these MPA treated stromal cell cultures to support long-term hemopoiesis was tested by seeding them with fresh bone marrow cells that had been passed through nylon wool. This procedure yields a relatively stromal cell depleted population of hemopoietic cells. An aliquot of 5 X 10(5) or 2.5 X 10(5) nylon wool passed bone marrow cells bearing the T6 chromosomal marker was seeded onto replicate MPA-treated stromal cell layers. The stromal cells stimulated the proliferation of the bone marrow cells, and nonadherent cells were present for up to 8 weeks of culture. Progenitors of granulocytes and macrophages (CFU-GM) were also present for this period of time despite weekly demi-depopulation, during culture feeding. Karyotypic analysis confirmed that the CFU-GM were derived from the reseeded population. Nylon wool-passed bone marrow cells seeded alone into empty flasks under identical conditions did not survive past 1 week. Cells from the reseeded cultures were also tested for early myeloid precursors (CFU-S) and injected into immunodeficient CBA/N mice to test for the presence of primitive B cell precursors. CFU-S were present in mice killed 11 days following injection of cells, and high levels of B cell colony-forming units (CFU-B) were present in mice 4 weeks post reconstitution. Further studies demonstrated that factors present in medium conditioned by the stromal cells could support the growth of CFU-GM. These data indicate that treatment of long-term bone marrow cultures with MPA results in a population of functional stromal cells.     

Dendritic cells from Chlamydia-infected mice show altered Toll-like receptor expression and play a crucial role in inhibition of allergic responses to ovalbumin

Our previous study has shown that Chlamydia lung infection can inhibit local eosinophilic inflammation induced by allergen sensitization and challenge, which is correlated with altered cytokine production. In the present study, we examined the role played by dendritic cells (DC) in chlamydial infection-mediated modulation of allergic responses. The results showed that DC freshly isolated from Chlamydia-infected mice (iIDC), unlike those from naive control mice (iNDC), could efficiently modulate immune responses to ovalbumin in vitro and in vivo. Co-culture of freshly isolated DC with naive CD4 cells from T cell receptor transgenic mice (DO11.10) showed that iIDC directed Th1-dominant, while iNDC directed Th2-dominant, allergen-specific CD4 T cell responses. Moreover, adoptive transfer of iIDC, but not iNDC, could inhibit systemic and local eosinophilia induced by allergen exposure. The reduction of eosinophilia was associated with a decrease in IL-5 receptor expression on bone marrow cells and the production of IL-5 and IL-13 by T lymphocytes. Analysis of the DC showed that iIDC expressed significantly higher levels of mRNA for Toll-like receptor 9 and produced more IL-12 compared to iNDC. The data demonstrate a critical role played by DC in infection-mediated inhibition of allergic responses.     

A subfraction of B220(+) cells in murine bone marrow and spleen does not belong to the B cell lineage but has dendritic cell characteristics

Although CD45R/B220 is commonly used as a pan-B cell marker in the mouse, not all B220(+) cells belong to the B cell lineage. Here we report the characterization of a subpopulation of B220(+)CD19(-) cells in murine bone marrow, which failed to express markers that are present in early CD19(--) B cell precursors. Instead, these cells expressed low levels of MHC class II and CD11c, which are typically found on dendritic cells (DC). Moreover, these B220(+)CD19(-)CD11c(+) cells expressed Gr-1, indicating that they are related to the recently identified murine plasmacytoid DC or their progenitors. Therefore, we evaluated surface marker expression of the B220(+)CD19(-)CD11c(+) cells in lymphoid tissues of C57BL/6 mice, recombinase activating gene-1 deficient mice, lacking mature B and T lymphocytes, and mice with a targeted disruption of the Ig H chain mu membrane exon (mu MT), lacking mature B lymphocytes. When comparing bone marrow and spleen, we found that the surface profiles of B220(+)CD19(-)CD11c(+) cells were remarkably similar, indicating that they are in a comparable maturation or activation stage in the two lymphoid compartments. In addition, the almost complete absence of peripheral B220(+) B-lineage cells in mu MT mice allowed the anatomical localization of the B220(+)CD19(-)CD11c(+) cells to the red pulp and the T cell areas in the spleen. Taken together, our findings indicate that the mouse bone marrow contains a recirculating population of B220(+)CD19(-) CD11c(+) plasmacytoid DC, the development of which is largely independent of the presence of mature T and B cells.