CD103 Dendritic Cells Research Articles

CD4+ T Cells Generated De Novo from Donor Hemopoietic Stem Cells Mediate the Evolution from Acute to Chronic Graft-versus-Host Disease

Acute and chronic graft-versus-host disease (GVHD) remain the major complications limiting the efficacy of allogeneic hemopoietic stem cell transplantation. Chronic GVHD can evolve from acute GVHD, or in some cases may overlap with acute GVHD, but how acute GVHD evolves to chronic GVHD is unknown. In this study, in a classical CD8+ T cell-dependent mouse model, we found that pathogenic donor CD4+ T cells developed from engrafted hemopoietic stem cells (HSCs) in C57BL/6SJL(B6/SJL, H-2(b)) mice suffering from acute GVHD after receiving donor CD8+ T cells and HSCs from C3H.SW mice (H-2(b)). These CD4+ T cells were activated, infiltrated into GVHD target tissues, and produced high levels of IFN-gamma. These in vivo-generated CD4+ T cells caused lesions characteristic of chronic GVHD when adoptively transferred into secondary allogeneic recipients and also caused GVHD when administered into autologous C3H.SW recipients. The in vivo generation of pathogenic CD4+ T cells from engrafted donor HSCs was thymopoiesis dependent. Keratinocyte growth factor treatment improved the reconstitution of recipient thymic dendritic cells in CD8+ T cell-repleted allogeneic hemopoietic stem cell transplantation and prevented the development of pathogenic donor CD4+ T cells. These results suggest that de novo-generated donor CD4+ T cells, arising during acute graft-versus-host reactions, are key contributors to the evolution from acute to chronic GVHD. Preventing or limiting thymic damage may directly ameliorate chronic GVHD.

CD44v7 ligation downregulates the inflammatory immune response in Crohn's disease patients by apoptosis induction in mononuclear cells from the lamina propria

Deletion of exon CD44v7 abrogates experimental colitis by apoptosis induction in intestinal mononuclear cells. Here we show that CD44v7 expression was upregulated upon CD40 ligation in human mononuclear cells, and examined whether ligation of CD44v7 also affects activation and apoptosis in lamina propria mononuclear cells (LPMC) from Crohn's disease (CD) patients. Thirty five patients with chronic inflammatory bowel disease (IBD), fourteen controls and four patients with diverticulitis were evaluated. CD44v7 was upregulated predominantly in the inflamed mucosa of CD patients. Furthermore, incubation with an anti-CD44v7 antibody induced apoptosis in LPMC isolated from inflamed mucosa of CD patients, but not from non-inflamed mucosa, from patients with ulcerative colitis (UC) or from normal controls. CD40 ligation and simultaneous incubation with anti-CD44v7 significantly downregulated CD80 in dendritic cells, thus inhibiting a critical second signal for naive T-cell activation. The apoptotic signal was mediated via the intrinsic mitochondrial pathway with decreased Bcl-2 and increased 7A6 (a mitochondrial membrane protein) expression. It was Fas independent and required caspases-3 and -9 activation. The process is highly specific for macrophage activation via CD40. These findings point to a novel mechanism of apoptosis induction in CD patients mediated by CD44v7 ligation.

Immune modulation through silencing CD80 and CD86 in dendritic cells using siRNA (88.4)

Abstract Dendritic cells (DC) are central cells of the immune system that can stimulate or suppress immune responses, partially depending on their expression of costimulatory molecules. CD80 and CD86 are critical costimulatory molecules for both T cell responses and Th differentiation. Thus, silencing DC expression of CD80 and CD86, using siRNA, could potentially enable manipulation of immune responses. Since silencing of CD80 or CD86 in DC has not been reported, in this study we investigated the tolerogenesis and Th differentiation capacity of CD80/CD86 in DC after siRNA-mediated gene silencing. Gene silencing was performed by transfecting mouse bone marrow-derived DC with CD80 or CD86 siRNA. The gene silencing efficacy was measured by flow cytometry, RT-PCR, and real time PCR. DC that were transfected with CD80 or CD86 siRNA induced inhibition of T cell responses in MLR and generated T regulatory cells. The CD4+CD25+FoxP+ cell subset was significantly increased in a co-culture system that contained CD80- or CD86-silenced DC and allogeneic T cells. Furthermore, CD80 siRNA significantly decreased IFN-gamma, although a reduction in IL-4 was not observed. Conversely, CD86 siRNA significantly reduced IL-4 but did not reduce IFN-gamma. This study suggested, for the first time, that CD80 siRNA and CD86 siRNA can be useful tools for generating tolerogenic DC with a differential effect on modulating cytokine production.

Efficient generation of CD34+ progenitor-derived dendritic cells from G-CSF-mobilized peripheral mononuclear cells does not require hematopoietic stem cell enrichment

As a result of their potent antigen-presentation function, dendritic cells (DC) are important tools for cell therapy programs. In vitro-generated DC from enriched CD34+ hematopoietic stem cells (HSC; enriched CD34 DC) have already proven their efficiency in Phase I/II clinical trials. Here, we investigated whether enrichment of CD34+ HSC before the onset of culture was absolutely required for their differentiation into DC. With this aim, we developed a new two-step culture method. PBMC harvested from G-CSF-mobilized, healthy patients were expanded for 7 days during the first step, with early acting cytokines, such as stem cell factor, fetal liver tyrosine kinase 3 ligand (Flt-3L), and thrombopoietin. During the second step, expanded cells were then induced to differentiate into mature DC in the presence of GM-CSF, Flt-3L, and TNF-alpha for 8 days, followed by LPS exposure for 2 additional days. Our results showed that the rate of CD34+/CD38+/lineageneg cells increased 19.5+/-10-fold (mean+/-sd) during the first step, and the expression of CD14, CD1a, CD86, CD80, and CD83 molecules was up-regulated markedly following the second step. When compared with DC generated from enriched CD34+ cells, which were expanded for 7 days before differentiation, DC derived from nonenriched peripheral blood stem cells showed a similar phenotye but higher yields of production. Accordingly, the allogeneic stimulatory capacity of the two-step-cultured DC was as at least as efficient as that of enriched CD34 DC. In conclusion, we report herein a new two-step culture method that leads to high yields of mature DC without any need of CD34+ HSC enrichment.

Differential Antigen Processing by Dendritic Cell Subsets in Vivo

Dendritic cells (DCs) process and present self and foreign antigens to induce tolerance or immunity. In vitro models suggest that induction of immunity is controlled by regulating the presentation of antigen, but little is known about how DCs control antigen presentation in vivo. To examine antigen processing and presentation in vivo, we specifically targeted antigens to two major subsets of DCs by using chimeric monoclonal antibodies. Unlike CD8+ DCs that express the cell surface protein CD205, CD8- DCs, which are positive for the 33D1 antigen, are specialized for presentation on major histocompatibility complex (MHC) class II. This difference in antigen processing is intrinsic to the DC subsets and is associated with increased expression of proteins involved in MHC processing.

Monocyte-derived DC Primed With TLR Agonists Secrete IL-12p70 in a CD40-dependent Manner Under Hyperthermic Conditions

Fever is an evolutionarily conserved mechanism to improve survival during infection. Previous studies have shown that feverlike temperatures directly enhance the function of murine bone marrow-derived dendritic cells (DCs). In the present study, we examined the response of human monocyte-derived DC to 39.5 degrees C hyperthermia. When primed with toll-like receptor agonists or bacterial extract but not proinflammatory cytokines, hyperthermia specifically enhanced secretion of interleukin (IL)-12p70 by DC, without altering the secretion of IL-10, tumor necrosis factor alpha or IL-1beta. These DC induced significantly higher levels of T-cell proliferation and interferon gamma production in assays of antigen presentation and MLR. Endogenous heat-sock protein 70 colocalized with CD40 in DC exposed to hyperthermic conditions. Recombinant CD40-Fc fusion protein blocked the increase in IL-12p70 secretion by DC primed with bacterial extract and hyperthermia. Thus, DC primed with toll-like receptor-agonists respond to hyperthermia with increased IL-12p70 secretion, mediated by heat-shock protein binding and activation of CD40. The data have important applications for clinical immunotherapy and the mechanism of fever.

TRACP Influences Th1 Pathways by Affecting Dendritic Cell Function

TRACP, a marker of osteoclasts, is also expressed by cells of the immune system. We identified a novel function for TRACP in the dendritic cell. DCs from TRACP knockout mice have impaired maturation and trigger reduced Th1 responses in vivo. We postulate that TRACP has an important role in the presentation of antigens to T cells. TRACP is highly expressed by osteoclasts, activated macrophages, and dendritic cells (DCs). Knockout mice lacking TRACP have an intrinsic defect in osteoclastic resorption and macrophages that display abnormal immunomodulatory responses and cytokine secretion profiles. Our aim in this study was to investigate the significance of TRACP in the inductive phase of the immune response by examining dendritic cells from TRACP(-/-) mice. Maturational state and function of leukocyte subsets in mice was assessed by flow cytometry. The ability of the immune system to respond to nonspecific activation and to specific antigen was assessed by delayed type hypersensitivity and the presence of isotype-specific serum antibody in vivo and T-cell proliferation and cytokine production in vitro. The ability of lipopolysaccharide (LPS) to upregulate MHC II and CD80 in DCs from TRACP(-/-) mice was reduced compared with wildtype mice, although production of IL-10 by DCs from TRACP-deficient animals was increased. T- and B-cell responses not involving antigen presentation (anti-CD3, TNP-ficoll) were normal in TRACP(-/-) mice, but responses to T-dependent antigens were impaired. Specifically, TRACP(-/-) mice had defective delayed hypersensitivity responses to picryl chloride and reduced proliferative responses to ovalbumin compared with wildtype mice. In response to ovalbumin, but not anti-CD3, T cells from TRACP(-/-) mice produced less interferon-gamma (IFN-gamma), but there was no difference in IL-4 production: TRACP(-/-) mice also produced less ovalbumin (OVA)-specific IgG2a after immunization. The finding that DCs from TRACP(-/-) mice have impaired maturation and defective Th1 responses shows that TRACP is important for polarizing responses in naïve T cells to antigen-presented dendritic cells.

Dendritic cells of immune thrombocytopenic purpura (ITP) show increased capacity to present apoptotic platelets to T lymphocytes

Altered self-antigen processing/presentation of apoptotic cells by DCs and/or modifications of autoantigens may lead to the development of autoantibodies. Increasing evidence indicates that platelets may undergo apoptosis. Therefore, in the present study we investigated whether platelet apoptosis and/or dendritic cells (DCs) may play a role in the stimulation of the immuno-mediated anti-platelet response in chronic immune thrombocytopenic purpura (ITP). Twenty-nine patients with active ITP and 29 healthy adult volunteers were enrolled into the study. Freshly washed platelets and platelets aged in a plasma-free buffer for 72 hours at 37 degrees C were assessed by flow cytometry for phosphatidylserine exposure using annexin V-FITC, caspase activation, and platelet activation markers. CD14-derived DCs were characterized by immunophenotyping, cytokine production, and ability to present fresh and aged platelets to T lymphocytes. We demonstrated that platelets from ITP patients, either fresh or in vitro aged, show increased apoptosis (with low levels of activation) in comparison to their normal counterparts. We also found that immature DCs readily ingest apoptotic platelets. Furthermore, in ITP patients DCs, prepulsed with autologous/allogeneic fresh and aged platelets, are highly efficient in stimulating autologous T-cell proliferation as compared to DCs derived from healthy donors. This finding may be related to the upregulated expression of CD86 in DCs from ITP patients and not to higher phagocytic activity. These results suggest that DC dysfunction, together with increased propensity of platelets to undergo apoptosis, may play a role in the stimulation of the immune system in ITP.

Cytotoxic T lymphocyte antigen‐4‐dependent down‐modulation of costimulatory molecules on dendritic cells in CD4+ CD25+ regulatory T‐cell‐mediated suppression

We have previously demonstrated that CD4+ CD25+ natural regulatory T cells (Treg cells) induce down-modulation of CD80 and CD86 (B7) molecules on dendritic cells (DCs) in vitro. In this report we show that the extent of down-modulation is functionally significant because Treg-cell conditioned DCs induced poor T-cell proliferation responses. Further, we report that down-modulation was induced rapidly and was inhibited by blocking cytotoxic T lymphocyte antigen-4 (CTLA-4), which is constitutively expressed by the Treg cells. Even though Treg cells have previously been reported to kill antigen-presenting cells, the down-modulation was not due to selective killing of DCs expressing high level of the costimulatory molecules. We propose that Treg cells down-modulate B7-molecules on DCs in a CTLA-4-dependent way, thereby enhancing suppression of T-cell activity.

Functional role of dendritic cells in patients with unstable angina

To investigate the function of dendritic cells (DC) in patients with unstable angina, 10 mL of blood was drawn from 30 subjects. 15 patients diagnosed as having unstable angina and 15 healthy subjects were included in an observation and a control groups respectively. The mononuclear cells were separated from the peripheral blood and cultured in RPM11640 supplemented with recombinant human granulocyte/macrophage-colony stimulating factor (rh GM-CSF) and recombinant human interleukin-4 (rh IL-4) to induce dendritic cells. The shape and ultrastructure of DC was examined with electronic microscope. The phenotype of DC was analyzed with FACS and the alloantigen presenting capacity of DC was evaluated by mixed lymphocyte reaction (MLR). The expression rate of CD86 of DC in patients with unstable angina was (40.7 +/- 3.6) %, which was obviously higher than that of normal DC (29.6 +/- 2.5%) ( P < 0.001). The capacity of the DCs in unstable angina patients to induce allogenic T cells (OD 2.73 +/- 1.10), was significantly higher than that of the normal DC (OD:0.9 +/- 0.21) (P < 0.005). It is suggested that the function of DC in patients with unstable angina is increased, which may play an important role in the initiation of immune reaction in the plaque.

Cognate CD4(+) T cell licensing of dendritic cells in CD8(+) T cell immunity.

Several studies have indicated that CD8(+) T cells require CD4(+) T cell help for memory formation. Evidence suggests that such help can be antigen independent, challenging whether the 'licensing' of dendritic cells (DCs) by CD4(+) T cells is ever required for cytotoxic T lymphocyte (CTL) responses. We show here that help is essential for the generation of CTL immunity to herpes simplex virus 1 and that CD4(+) T cells mediate help in a cognate, antigen-specific way. We provide direct in vivo evidence for DC licensing by helper T cells and show that licensing is rapid and essential for the formation of effector and memory CTLs. In situations in which DCs are poorly licensed by pathogen-derived signals, our findings suggest that CTL immunity may be heavily dependent on cognate DC licensing.

Administration of dendritic cells transduced with antisense oligodeoxyribonucleotides targeting CD80 or CD86 prolongs allograft survival.

The expression of costimulatory molecules on antigen-presenting cells is crucial in determining T-cell immune responses. We examined the effects of transduction with high-affinity antisense oligodeoxyribonucleotides (ODNs) designed to target the mRNA of CD80 or CD86 on the phenotype and function of dendritic cells (DCs). DCs were propagated from C57BL/10 (B10; H2b) bone marrow cells in granulocyte macrophage-colony stimulating factor and interleukin (IL)-4, and transduced with anti-CD80 or anti-CD86 antisense ODNs (base-mismatched ODNs as controls). The effect of antisense ODN on phenotype was examined by flow cytometry. The allostimulatory function of DCs was assessed by mixed leukocyte reaction and cytotoxic activity in vitro, and the influence on allograft survival was assessed in vivo. ODNs were effectively incorporated by DCs, which were enhanced by the presence of lipofectamine. Antisense ODNs targeting CD80 or CD86 mRNA specifically suppressed the expression of CD80 or CD86 in DCs and inhibited their capacity to elicit the proliferative responses, donor-specific cytotoxic T-lymphocyte activity in C3H (H2k) spleen T cells. This was associated with decreased IL-2, but elevated IL-4 production, and an increase in T-cell apoptosis. In contrast with the administration of control DCs into C3H recipients that exacerbated rejection of B10 cardiac allografts, injection of DCs transduced with anti-CD80 or CD86 antisense ODN significantly prolonged the survival of heart allografts. Transduction with antisense ODN targeting CD80 or CD86mRNA is a feasible and effective approach to modify DCs that renders them tolerogenic by inducing T-cell hyporesponsiveness and apoptosis. This may lead to the development of new therapeutic strategies in transplantation.

The importance of CD11b+ dendritic cells in CD4+ T cell activation in vivo: with help from interleukin 1.

A murine model of cutaneous leishmaniasis, using needle inoculation of Leishmania major, represents perhaps the most widely used system to study the contrasting effects of Th1 versus Th2 immunity in disease control. One of the intriguing aspects of this disease model is that certain strains of mice

CTL priming by CD8+ and CD8 dendritic cells in vivo

Two distinct developmental pathways are driving the formation of myeloid- and lymphoid-related dendritic cells (DC) which differ in anatomical localization and phenotype. In terms of function, it has been hypothesized that only the myeloid-related CD8– DC are able to initiate immune responses, whereas the lymphoid-related CD8+ DC have been suggested to induce tolerance. Here we show that both subsets activate CD8+ T cells in vitro and induce protective anti-viral CTL responses in vivo. Thus, vaccine strategies using peptide-pulsed DC do not have to take into account DC subsets for priming.

CpG DNA: a potent signal for growth, activation, and maturation of human dendritic cells.

DNA molecules containing unmethylated CpG-dinucleotides in particular base contexts ("CpG motifs") are excellent adjuvants in rodents, but their effects on human cells have been less clear. Dendritic cells (DCs) form the link between the innate and the acquired immune system and may influence the balance between T helper 1 (Th1) and Th2 immune responses. We evaluated the effects of CpG oligodeoxynucleotides alone or in combination with granulocyte-macrophage colony-stimulating factor (GMCSF) on different classes of purified human DCs. For primary dendritic precursor cells isolated from human blood, CpG oligonucleotides alone were superior to GMCSF in promoting survival and maturation (CD83 expression) as well as expression of class II MHC and the costimulatory molecules CD40, CD54, and CD86 of DCs. Both CD4-positive and CD4-negative peripheral blood dendritic precursor cells responded to CpG DNA which synergized with GMCSF but these DCs showed little response to lipopolysaccharide (LPS). In contrast, monocyte-derived DCs did not respond to CpG, but they were highly sensitive to LPS, suggesting an inverse correlation between CpG and LPS sensitivity in different subsets of DCs. Compared with GMCSF, CpG-treated peripheral blood DCs showed enhanced functional activity in the mixed lymphocyte reaction and induced T cells to secrete increased levels of Th1 cytokines. These findings demonstrate the ability of specific CpG motifs to strongly activate certain subsets of human DCs to promote Th1-like immune responses, and support the use of CpG DNA-based trials for immunotherapy against cancer, allergy, and infectious diseases.

HLA-G in the human thymus: a subpopulation of medullary epithelial but not CD83(+) dendritic cells expresses HLA-G as a membrane-bound and soluble protein.

The human MHC class Ib gene HLA-G is transcribed and translated in different placental cell subpopulations during pregnancy. In addition to this restricted tissue distribution, HLA-G proteins were also recently detected in the thymus of HLA-G transgenic mice, as well as in some human thymic epithelial cells (TEC). There was a need to further define the phenotype of the HLA-G-expressing cells in the human thymus as well as the type of translated forms that they produce. Using several HLA-G-specific mAb and immunohistochemistry performed on cryosections of human thymi at different ages, we found that the HLA-G-expressing cells are present on medullary cells exhibiting the epithelial morphological type 6. Co-localization experiments performed by double or triple immunofluorescence staining demonstrate that these HLA-G-expressing cells express various cytokeratins, epithelial cell markers but not the CD83 dendritic cell marker. We further show by ELISA measurements that a subset of primary cultured human TEC also expresses soluble HLA-G. Therefore, HLA-G protein tissue distribution is not restricted solely to placental cells. A subpopulation of medullary TEC also expresses HLA-G both at their cell surface and in secreted form, raising the question of the functional significance of such MHC class Ib molecules. Whether thymic soluble and/or membrane-bound HLA-G contribute to inhibit NK cells or to a negative selection of autoreactive T cells which could be harmful in case of pregnancy and/or to a positive selection of viral peptides/HLA-G-restricted CD8(+) T cells remains to be demonstrated.