The distribution and functional properties of dendritic cells in patients with seronegative arthritis.

Dendritic cells (DC), potent antigen-presenting cells, are known to be increased in numbers in inflammatory lesions in rheumatoid arthritis and juvenile chronic arthritis. In this study, patients with seronegative arthritis were studied; the distribution and functional properties of DC enriched low density cells (LDC) from peripheral blood (PB) and synovial fluid (SF) were compared. The composition of LDC from both sources was similar, comprising approximately 30% DC, 60% monocytes with few T lymphocytes. SF was significantly enriched for LDC compared with paired peripheral blood (P less than 0.0001) or peripheral blood from healthy controls (P less than 0.001). In contrast, patient PB contained fewer LDC (P less than 0.05) overall than healthy controls. LDC from both sources were potent simulators of allogeneic PB T cells in a mixed leucocyte reaction (MLR), but in four out of 10 patients SF LDC were significantly more stimulatory. In autologous MLRs (AMLRs) SF T cells were not stimulated by either LDC population. This anergy of T cells was confined to the joint as patient PB T cells showed an AMLR response to PB LDC which was similar to that seen in cells from healthy controls. PB T cells also responded to SF LDC; in a minority of patients SF LDC caused significantly greater stimulation in AMLR than PB LDC and the possibility is discussed that this may represent presentation of antigen acquired in vivo.     

During HIV-1 infection most blood dendritic cells are not productively infected and can induce allogeneic CD4+ T cells clonal expansion.

We have considered the possibility that antigen-presenting cells of the dendritic cell lineage may be infected in vivo and spread HIV-1 at the time dendritic cells initiate the clonal expansion of antigen-specific T cells. Dendritic cells were isolated from 25 HIV-1-infected subjects (CDC stages II-IV). Fewer dendritic cells were recovered from most infected subjects. Reduced numbers of total non-T cells were also found in these patients, so that preferential loss of dendritic cells did not occur. Dendritic cell function was assessed by stimulatory capacity for allogeneic CD4+ T cells in the mixed leucocyte reaction (MLR). Potent MLR stimulator activity was retained in the dendritic cell-enriched populations from HIV-infected patients. Seven out of nine patients without AIDS (asymptomatic, lymphadenopathy or ARC) and three out of six patients with AIDS had proliferative responses equivalent to those induced by dendritic cells from controls. Dendritic cells from HIV+ subjects were able to initiate the expansion of allogeneic CD4+ T cell clones with cloning efficiency not different from controls and without evidence of cytopathic effect in the expanding CD4+ clones. In situ hybridization of the different mononuclear cell populations with a gag-specific riboprobe demonstrated positive cells in the T cell fractions of 12 of the 15 patients tested. None of the asymptomatic or ARC patients had riboprobe-positive cells in the dendritic cell-enriched populations. Four out of nine patients with AIDS had cells positive for HIV-1 expression in the dendritic cell-enriched fraction. However, the positive cells had the nuclear profile of lymphocytes, and by cytofluorography some residual low-density T cells were present. By limiting dilution and polymerase chain reaction (PCR), CD4+ lymphocytes carried HIV provirus in inocula of 500-5000 cells, while provirus could only be detected in 50,000 cells from the dendritic cell-enriched fraction. The latter signal may be due to the demonstrated levels of T cell contamination. Our data indicate that productive or latent HIV-1 infection of blood dendritic cells in vivo is rare, certainly no greater than in T lymphocytes, and that in vitro dendritic cell preparations from patients can expand CD4+ T cells efficiently and therefore may be able to expand T cells with immunotherapeutic activity.     

Glucocorticoids modulate the development of dendritic cells from blood precursors

Dendritic cells (DC) are professional antigen-presenting cells, capable of priming naive T cell responses. Glucocorticoids (GC) are frequently used in asthmatic patients. In this study we describe the effects of GC on the development and function of monocyte-derived DC (MoDC) in vitro and in vivo. Monocytes from healthy individuals were isolated and incubated with granulocyte-macrophage colony-stimulating factor (GM-CSF) and IL-4 for 6 days, to induce maturation into MoDC. To study the role of GC on DC differentiation in vitro cells were incubated with dexamethasone at different stages of MoDC development. At day 6 cells were characterized phenotypically by flow cytometry and functionally in an allogeneic mixed leucocyte reaction. To study the effect of GC in vivo patients with mild/moderate atopic asthma were selected. In one group no GC were used, whereas the other group used inhalation GC. MoDC from these patients were generated as described above and tested functionally. Incubation of MoDC or its peripheral blood precursors with dexamethasone decreased the accessory potency dose-dependently. The functional differences could not be explained by the changes in the expression of MHC II and the costimulatory molecules CD40 and CD86. The relevance of this mechanism was confirmed for the in vivo situation as well. MoDC from patients using inhalation GC showed a decreased accessory potency. These data suggest a modulatory effect of GC therapy at the level of the peripheral blood monocyte. The results indicate that GC influence DC development and function in vitro as well as in vivo.     

Enrichment and characterization of dendritic cells from human bronchoalveolar lavages.

In the present study about 0.3% to 1.6% of human bronchoalveolar lavage (BAL) cells were identified as typical dendritic cells (DC), having an irregular outline, lobulated nucleus, and clear distinguishable acid phosphatase activity or EBM11 (anti-CD68) reactivity in a spot near the nucleus. After DC enrichment, using transient adherence to plastic, FcR-panning, and a density metrizamide gradient, a population containing 7-8% typical DC was obtained. This DC-enriched low density fraction, containing the highest percentages of DC, very strongly induced T cell proliferation in an allogeneic mixed leucocyte reaction (MLR), which was significantly higher than that induced by other partly (un)fractionated BAL cells. These data indicate that DC seem to be the major accessory cells in the BAL fluid, and therefore may be important in the regulation of T cell immune responses in the lung.     

An approach to isolating T cell lines that react to antigens presented on the surface of dendritic cells.

We describe an approach that might be useful for identifying antigens on surfaces of antigen presenting cells. It is known that dendritic cells carry antigens in situ and are efficient at clustering antigen-specific T cells. Using the human mixed lymphocyte reaction (MLR) system, we have shown that alloreactive CD4+ T cells can be selected by their capacity to cluster with dendritic cells in the first 2 days of the MLR. Small numbers of clustered cells, 1-10/culture well, could then be expanded as antigen-specific lines in presence of either antigen or mitogen, sodium periodate. Few antigen-specific lines could be isolated from the nonclustered fraction. When T cell lines derived from the dendritic T cell clusters were maintained without antigen, i.e. using second party (syngeneic antigen-presenting cells (APC] or irrelevant antigen bearing APC, i.e. third-party (HLA-mismatched) stimulator cells plus mitogen, the T cells retained their specificity for the original stimulating alloantigen over the time course tested, several weeks to months. These findings show that by using dendritic cells as immunoadsorbents one can prepare antigen-specific cell lines and maintain the specificity of the lines without the need for adding exogeneous antigen during either immunoselection or cloning. We discuss the possible use of dendritic cells as a means for raising T cell lines and clones that recognize antigens being carried by APC and which might be pertinent to protective immunity and autoimmunity.     

Dendritic cells derived from bone marrow cells fail to acquire and present major histocompatibility complex antigens from other dendritic cells

Dendritic cells stimulate primary T-cell responses and a major activation route is via presentation of antigens pre-processed by other dendritic cells. This presentation of pre-processed antigens most likely proceeds through transfer of functional major histocompatibility complex (MHC) antigens through exosomes, 'live nibbling' or apoptotic vesicles. We hypothesized that not all dendritic cell populations may both donate MHC antigen to dendritic cells and present antigens acquired from other dendritic cells. All populations tested, including those derived from bone marrow precursor cells stimulated primary, allogeneic T-cell responses and acted as accessory cells for mitogen stimulation. Populations of responder type, splenic dendritic cells promoted allogeneic responses indirectly but those derived from bone marrow cells blocked rather than promoted T-cell proliferation. To identify mechanisms underlying this difference we studied transfer of I-A antigens between cells. Active, two-way transfer of allogeneic I-A occurred between splenic primary antigen presenting cells including CD8alpha+ lymphoid dendritic cells, CD8alpha- myeloid dendritic cells and B220+ cells; all these cell types donated as well as acquired MHC molecules. By contrast, the bone marrow-derived dendritic cells donated I-A antigens but acquired negligible amounts. Thus, dendritic cells derived directly from bone marrow cells may stimulate primary T-cell responses through transferring functional MHC to other dendritic cells but may not be able to acquire and present antigens from other dendritic cells. The evidence suggests that T-cell activation may be blocked by the presence of dendritic cells that have not matured through lymphoid tissues which are unable to acquire and present antigens pre-processed by other dendritic cells.     

Isolation and characterization of dendritic cells from adenoids of children with otitis media with effusion.

Dendritic cells were enriched from adenoids of children with otitis media with effusion (OME) by density gradient centrifugation and culture techniques. An enrichment of 40-140-fold was obtained for dendritic cells. These cells were identified using morphology, enzyme cytochemistry, immunocytochemistry and functional criteria. Dendritic cells could be easily distinguished from macrophages. It appeared that the MoAb EBM11 (CD68) discriminated between dendritic cells and macrophages; in dendritic cells this activity was localized in a spot, whereas in macrophages it was found throughout the whole cytoplasm. The fractions enriched with dendritic cells showed a strong stimulatory effect on allogeneic T cells. These responses were MHC class II dependent since they could be blocked by anti-HLA-DR/DQ MoAbs. The data clearly show that dendritic cells from adenoids of children with OME still have functional capacities.     

Autologous apoptotic T cells interact with dendritic cells,but do not affect their surface phenotype or their ability to induce recall immune responses

Dendritic cells (DCs) play an important role in determining immunogenicity and the subsequent immune response. They may also have a role in maintaining peripheral tolerance to self-antigens by initiating an immune response only in the context of danger signals released from cells during stress, damage or death. These signals may originate from surrounding T cells as well as from other cells. Therefore, in this study the effect of autologous T cell injury on DC morphology and function has been investigated. Co-incubation of apoptotic or necrotic T cells with immature DCs altered their morphology towards a more mature appearance, with more cells showing activation as judged by spreading and formation of arborizing long processes. The apoptotic autologous T cells were rarely phagocytosed by immature DCs, compared to macrophages. The DC surface phenotype was not affected by the co-incubation with autologous injured T cells. The ability of DCs to elicit a secondary immune response was not altered by exposure to autologous injured T cells. These findings suggest that DC can continue to function in T cell activation, rather than in tolerogenic mode, even in the presence of large numbers of dying autologous T cells, such as may be present in the aftermath of an acute antiviral response.     

Dendritic cells infected in vitro with human T cell leukaemia/lymphoma virus type-1 (HTLV-1); enhanced lymphocytic proliferation and tropical spastic paraparesis.

Evidence supporting a role of the dendritic cell (DC) in stimulating autologous T cell activity in tropical spastic paraparesis (TSP) was sought by studies of cells taken from healthy volunteers and exposed to HTLV-1 in vitro. DC were co-cultured with an HTLV-1-producing cell line (MT-2) at 1:1 or 10:1 ratios. These DC stimulated high levels of proliferation in autologous T cells. This was similar to that seen in an autologous mixed leucocyte reaction (AMLR) using cells from TSP patients. The requirement for both DC and virus was confirmed, since neither DC co-cultured with uninfected MT-2 cells nor addition of infected MT-2 cells directly to T cells caused significant stimulation. DC exposed to the highest dose of HTLV-1 (1:1) for 24 h before addition of T cells caused strong stimulation that increased after 8 h but almost disappeared by 72 h. In situ hybridization showed that approximately 25% of DC became infected in cultured cells after preincubation for 24 h, and over 50% were infected with a 72-h preincubation. We suggest that infection of DC by HTLV-1 may be an initial step in altering the immune system in seronegative patients, and that persistent T cell stimulation in those with genetic susceptibility may underlie the production of neurological disease.     

Relationship between impaired apoptosis of lymphocytes and distribution of dendritic cells in peripheral blood and synovial fluid of children with juvenile idiopathic arthritis

INTRODUCTION: The pathogenesis of juvenile idiopathic arthritis (JIA) is not fully understood. Recently the present authors described disturbed apoptosis of JIA lymphocytes in both peripheral blood (PB) and synovial fluid (SF) as well as an abnormal distribution of blood dendritic cells (BDCs) between the PB and SF in this disease. Possible relationships between these events during the development of JIA process are assessed here. MATERIALS AND METHODS: Lymphocyte apoptosis and BDC counts were assessed in the PB and SF of untreated JIA children. Lymphocyte apoptosis was analyzed by the Annexin-V/propydium iodide assay. Total DC (TDC) number was based on the sum of three BDC subpopulations determined using a panel of monoclonal antibodies against BDC antigens (BDCA): myeloid type 1 (mDC1, BDCA-1(+)/HLA-DR(+)/CD19(-)), myeloid type 2 (mDC2, BDCA-3(+)/HLA-DR(+)/CD14(-)), and plasmacytoid (pDC, BDCA-2(+)/HLA-DR(+)/CD123(+)). Cells were enumerated by the flow cytometric "single-platform" method. The concentration of tumor necrosis factor (TNF)-alpha and the distribution of particular lymphocyte subtypes in both PB and SF were also investigated. RESULTS: There was significant positive correlation between apoptosis of PB lymphocytes and SF TDC count (p=0.002) as well as SF TNF-alpha concentration (p=0.007). SF TNF-alpha levels also correlated with SF TDC count (p=0.003). Moreover, JIA SF was distinctly enriched with CD4+ and CD8+ T lymphocytes and included CD4(+)/CD25(high) cells as well. There was significant positive correlation between the number of CD4(+)/CD25(high) cells and SF JIA BDC count (p=0.015). CONCLUSIONS: These data suggest a possible link between impaired apoptosis of PB/SF lymphocytes and increased recruitment of PB BDCs to SF and other elements of the immune system in JIA, including regulatory CD4+/CD25high cells.     

Calcium responses elicited in human T cells and dendritic cells by cell-cell interaction and soluble ligands.

The interactions between a human CD4+ T cell clone and monocyte-derived human dendritic cells (DC) were analyzed with an imaging system. The first question addressed was the relationship between the formation of a contact zone and the triggering of a Ca2+ response in the T cells, in the presence or absence of antigen. Interaction of T cells with DC pulsed with the antigen led to the formation of a stable contact zone, followed by the appearance in the T cells of large and sustained Ca2+ oscillations. In the absence of antigen, contact zones formed normally and, surprisingly, Ca2+ responses were also observed, characterized by rare and small transients. Antigen-independent Ca2+ responses were not MHC restricted. The possible influence of Ca2+ responses in the DC on the efficiency of antigen presentation was then Investigated. In DC, Ca2+ responses can be elicited by a variety of stimuli: cell adhesion, platelet-activating factor, UTP and chemotactic molecules (formyl-Met-Leu-Pro, RANTES, MIP-1beta and SDF-1alpha). Importantly, Ca2+ responses were also induced in approximately 30% of DC as a result of their interaction with T cells. However, the efficiency of antigen presentation (as judged by the percentage of T cells presenting a Ca2+ response) was independent of the Ca2+ level in DC. Thus, imaging the interactions between human T cells and DC led us to observe two novel phenomena: DC-induced but antigen-independent Ca2+ responses in T cells and T cell-induced Ca2+ responses in DC.     

妊娠特异性糖蛋白对人外周血树突状细胞的免疫调节作用

目的探讨妊娠特异性糖蛋白对人外周血树突状细胞（DC）的免疫调节作用。方法常规分离、培养外周血树突状细胞,一定浓度妊娠特异性糖蛋白作用后,采用三色抗体（Lin1-FITC、抗HLA-DR-Percp和PE标记的CD80、CD86、CD11c和CD123抗体）流式检测DC的表型;流式细胞仪检测DC摄取抗原PE-OVA的能力;MTT法检测DC刺激同种T淋巴细胞的增殖能力。结果与对照组比较,妊娠特异性糖蛋白作用后的DC膜表面分子CD80、CD86和CD11c的阳性百分率明显降低（P〈0.01）,而CD123的阳性率明显升高（P〈0.01）;妊娠特异性糖蛋白作用后的DC的抗原摄取能力及刺激同种T细胞的增殖能力均明显降低（P〈0.01）。结论妊娠特异性糖蛋白对外周血树突状细胞具有免疫下调作用。     

Differential response of human naive and memory/effector T cells to dendritic cells infected by respiratory syncytial virus

In vitro studies have contributed substantially to the understanding of immunopathology of respiratory syncytial virus (RSV)-mediated disease. In the present study we compared the effect of RSV-infected dendritic cells on the time-course of the primary and memory/effector T cell response in vitro. Cultures with uninfected dendritic cells known to elicit T helper 2 (Th2) responses and with polyinosinic-polycytidylic acid (poly-IC)-stimulated dendritic cells known to elicit Th1 responses served as controls. At day 1 after stimulation there was a high proportion of interleukin (IL)-2 and tumour necrosis factor (TNF)-alpha-producing T cells with no difference in number of producing T cells as well as concentration of secreted cytokines between RSV-infected and control cultures. However, up to day 3 generation of IFN-gamma was reduced markedly. In addition, there was a reduced proliferation in RSV cultures. At day 7 the RSV-treated cultures showed a preponderance of IL-4 generation. At days 21-24, after three rounds of restimulation, memory/effector T cells matured under the influence of RSV were still not fully polarized but in contrast to the primary response displayed a predominance of Th1 cytokines. Contact with RSV-infected HEp-2 cells inhibited proliferation of T cells; memory effector T cells were less sensitive to contact inhibition than naive T cells. In addition, RSV inhibited the stimulated rearrangement of cortical actin more effectively in naive compared to memory T cells. In summary, we have shown that RSV infection of dendritic cells has a distinct modulatory effect on the primary response and a less pronounced effect on the memory response.     

Impact of IgA isoforms on their ability to activate dendritic cells and to prime T cells

Human IgA could be from different isotypes (IgA1/IgA2) and/or isoforms (monomeric, dimeric, or secretory). Monomeric IgA mainly IgA1 are considered as an anti-inflammatory isotype whereas dimeric/secretory IgA have clearly dual pro- and anti-inflammatory effects. Here, we show that IgA isotypes and isoforms display different binding abilities to FcαRI, Dectin-1, DC-SIGN, and CD71 on monocyte-derived dendritic cells (moDC). We describe that IgA regulate the expression of their own receptors and trigger modulation of moDC maturation. We also demonstrate that dimeric IgA2 and IgA1 induce different inflammatory responses leading to cytotoxic CD8⁺ T cells activation. moDC stimulation by dimeric IgA2 was followed by a strong pro-inflammatory effect. Our study highlights differences regarding IgA isotypes and isoforms in the context of DC conditioning. Further investigations are needed on the activation of adaptive immunity by IgA in the context of microbiota/IgA complexes during antibody-mediated immune selection.     

A comparison between isolated blood dendritic cells and monocyte‐derived dendritic cells in pigs

Various dendritic cell (DC) populations exist that differ in phenotype and ability to present antigen to T cells. For example, plasmacytoid DCs (pDCs) are less potent T cell activators compared with conventional DCs (cDCs). Here, we compared porcine blood DCs (BDCs), containing pDCs and cDCs, and monocyte‐derived DCs (MoDC), consisting of cDCs, in their phenotype, ability to uptake antigen, activation and maturation and their ability to present antigen to autologous T cells. Pigs represent an important animal model, whose immune system in many respects closely resembles that of humans. For example, the distribution of Toll‐like receptors is similar to that of humans, in contrast to that of mice. Here we demonstrate that both populations endocytose foreign material. Following lipopolysaccharide stimulation, CD80/86 and chemokine receptor (CCR)7 expression was increased in both populations as was the expression of the chemokine ligands (CCL)‐2, CCL‐4, CCL‐20 and CXCL‐2. Although basal and post‐stimulation protein concentrations of interleukins 6 and 8 and tumour necrosis factor‐α were higher in MoDCs, protein concentrations showed a higher fold increase in BDCs. Antigen‐specific proliferation of autologous T cells was induced by MoDCs and BDCs. Interestingly, while MoDCs induced stronger proliferation in naive T cells, no difference in proliferation was observed when primed T cells were studied. These results demonstrate that isolated porcine BDCs are highly responsive to stimulation with lipopolysaccharide and are functionally able to drive primed T‐cell proliferation to the same extent as MoDCs.     

The proliferative response of CD4 T cells to steady-state CD8+ dendritic cells is restricted by post-activation death

CD8(+) splenic dendritic cells (DCs) from steady-state mice are less effective than the CD8(-) DC subset in their capacity to stimulate CD4 T cell proliferation in culture. However, we found that the two DC subtypes were equally potent at activating CD4 T cells, based on up-regulation of CD69 and CD25 expression. Also, we found no difference in the rate of T cell death prior to entry into the first division. We then tracked carboxyfluorescein diacetate succinimidyl ester-labeled T cells and employed a quantitative model to assess in detail the CD4 T cell expansion process in response to stimulation with CD8(+) or with CD8(-) DCs. The time required for most T cells to replicate their DNA prior to the first division was similar in both DC cultures. However, progression of the CD4 T cell population through subsequent divisions was reduced in CD8(+) DCs compared with CD8(-) DC culture. This was associated with an increased loss of viable T cells at each division. Post-activation, division-associated T cell death is therefore a major factor in the reduced response of CD4 T cells to CD8(+) DCs.     

Dexamethasone transforms lipopolysaccharide-stimulated human blood myeloid dendritic cells into myeloid dendritic cells that prime interleukin-10 production in T cells

Myeloid dendritic cells (MDC) play an important role in antigen-specific immunity and tolerance. In transplantation setting donor-derived MDC are a promising tool to realize donor-specific tolerance. Current protocols enable generation of tolerogenic donor MDC from human monocytes during 1-week cultures. However, for clinical application in transplantation medicine, a rapidly available source of tolerogenic MDC is desired. In this study we investigated whether primary human blood MDC could be transformed into tolerogenic MDC using dexamethasone (dex) and lipopolysaccharide (LPS). Human blood MDC were cultured with dex and subsequently matured with LPS in the presence or absence of dex. Activation of MDC with LPS after pretreatment with dex did not prevent maturation into immunostimulatory MDC. In contrast, simultaneous treatment with dex and LPS yielded tolerogenic MDC, that had a reduced expression of CD86 and CD83, that poorly stimulated allogeneic T-cell proliferation and production of T helper 1 (Th1) cytokines, and primed production of the immunoregulatory cytokine interleukin-10 (IL-10) in T cells. In vitro, however, these tolerogenic MDC did not induce permanent donor-specific hyporesponsiveness in T cells. Importantly, tolerogenic MDC obtained by LPS stimulation in the presence of dex did not convert into immunostimulatory MDC after subsequent activation with different maturation stimuli. In conclusion, these findings demonstrate that combined treatment with dex and LPS transforms primary human blood MDC into tolerogenic MDC that are impaired to stimulate Th1 cytokines, but strongly prime the production of the immunoregulatory cytokine IL-10 in T cells, and are resistant to maturation stimuli. This strategy enables rapid generation of tolerogenic donor-derived MDC for immunotherapy in clinical transplantation.