Dendritic cells: a conductor of T cell differentiation.

Induction of different types of adaptive immune responses depending on the nature of antigens and the environmental context is crucial to cope with a variety of pathogens and concurrently to avoid pathological reaction to self antigens. Recent studies have been elucidating that the diversity of immune responses is critically controlled by dendritic cells (DCs). Two DC subsets have been identified in humans: myeloid DCs and plasmacytoid DCs. The DC subsets induce different types of adaptive immune responses depending on environmental factors. Interleukin (IL)-12 from myeloid DCs is a dominant factor for the induction of a Th1 response, whereas OX40 ligand on myeloid DCs is important for the induction of a Th2 response. Furthermore, inducible costimulator (ICOS) ligand on plasmacytoid DCs is critical for the induction of IL-10-producing regulatory T cells. Elucidating cellular and molecular mechanisms by which functions of the two DC subsets are modulated will lead to understanding the pathogenesis of various immune-related diseases and to developing novel immunological therapies.     

小鼠巨细胞病毒感染鼠胚胎成纤维细胞对共培养调节性T细胞增殖和活化的影响

目的 研究小鼠巨细胞病毒(MCMV)感染对共培养体系中调节性T细胞(Treg)的诱导作用和Treg在抗病毒免疫中的调节作用.方法 建立小鼠T淋巴细胞与感染CMV的同系小鼠胚胎成纤维细胞(MEF)体外共培养的细胞模型.蚀斑法检测共培养上清液中MCMV负荷量;细胞计数法分析体系中T淋巴细胞的扩增;流式细胞术检测共培养体系中CD4+ CD25+ Treg细胞比例,Western印迹法检测T淋巴细胞中Foxp3蛋白表达强度;RT-PCR法检测MEF中转化生长因子(TGF)-β mRNA表达水平;双抗体夹心ELISA法检测细胞培养上清液中TGF-β蛋白表达水平.组间差异采用单因素方差分析.结果 T淋巴细胞与MCMV感染的MEF共培养1 d和3 d后,培养上清液中病毒负荷量较对照组显著减少;但是共培养6 d后,T淋巴细胞的免疫保护作用消失,病毒量为(5.58±0.67)×105 PFU/mL,与感染对照组的(6.05±0.34)×105 PFU/mL比较,差异无统计学意义.CMV感染3 d后,T淋巴细胞数由感染前的(2.02±0.05)×106 /mL 增至(2.25±0.13)×106 /mL (P＜0.05);但继续培养至6 d时,T淋巴细胞数为(2.08±0.14)× 106/mL,与3 d时比较,差异无统计学意义.CD4+ CD25+ Foxp3+ Treg细胞比例和Foxp3蛋白表达变化相似,均随MCMV感染时间延长而增加,在感染后6 d分别增至对照组的2倍和3倍;TGF-β在MEF中的基因转录水平(A值)由感染前的1.09±0.13增至感染3 d后的3.15±0.54,共培养上清液中的蛋白表达强度在感染后3 d增至(3.85±0.32)μg/L,与感染前的(1.74±0.14)μg/L比较,差异有统计学意义(P＜0.05).结论随感染时间延长,MCMV可能通过促进TGF-β表达等多种途径诱导Treg增殖活化.     

Thrombospondin from activated platelets promotes sickle erythrocyte adherence to human microvascular endothelium under physiologic flow: a potential role for platelet activation in sickle cell vaso-occlusion

Adherence of erythrocytes to vascular endothelium likely contributes to the pathophysiology of episodic vascular occlusion in patients with sickle cell disease (SCD). In addition, coagulation activation has been reported in sickle patients during complications such as pain episodes. To test the hypothesis that platelet activation contributes to sickle erythrocyte binding, we investigated whether factors released from activated sickle platelets promote adherence of sickle erythrocytes to human microvascular endothelial cells (MEC) under flow conditions. Activated sickle platelet supernatant (ASPS) promoted high levels of sickle erythrocyte adherence to MEC (55.4 +/- 3.9 erythrocytes/mm2) but only moderate adherence of normal erythrocytes to MEC (14.1 +/- 0.7 erythrocytes/mm2). When MEC were incubated with an antibody (OKM5) against CD36 (a thrombospondin [TSP] receptor), platelet supernatant mediated sickle erythrocyte adherence was inhibited 86%, suggesting that TSP participated in the adherence. To further define the role of TSP in adherence, additional studies using purified TSP were performed. At a concentration of 0.2 micrograms/mL TSP in serum-free media (SFM), sickle erythrocyte adherence to MEC was 33.9 +/- 2.7 erythrocytes/mm2 and sixfold greater than either sickle erythrocyte adherence in the absence of TSP or normal erythrocyte adherence in the presence of TSP. Doubling the concentration of TSP to 0.4 micrograms/mL proportionally increased adherence of sickle erythrocytes. Incubation of MEC with OKM5 or anti-alpha v monoclonal antibodies inhibited TSP-mediated sickle erythrocyte adherence more than 95%. These data suggest that activated platelet release factors, including alpha-granule TSP, which promote receptor-mediated sickle erythrocyte adherence to microvascular endothelium. Such factors released during in vivo platelet activation could contribute to vaso-occlusive complications by promoting erythrocyte adherence and microvascular occlusion.     

Transmission and activation of cytomegalovirus with blood transfusion: a mouse model.

A mouse model that mimics many features of human cytomegalovirus (CMV) infections associated with transfusion and perfusion is described. The concept of antigenic activation of CMV was tested by infusion of blood from latently infected mice, which were found to be virus-negative by tissue culture asssay, into uninfected allogeneic and isogenic hosts. After a latent period, virus was detectable invariably in allogeneic and only rarely in isogenic recipients. Transfusions from uninfected donors into latently infected mice activated CMV in heterologous and homologous recipients. These observations should assist in definition of relevant pathogenetic principles and may explain the failure to recover CMV from healthy human blood donors in spite of predictions of a carrier state based on epidemiologic observations.     

Dendritic cell and natural killer cell cross-talk: a pivotal role of CX3CL1 in NK cytoskeleton organization and activation

Initial molecular events leading to natural killer lymphocyte (NK) and dendritic cell (DC) interactions are largely unknown. Here, the role of CX3CL1 (fractalkine), a chemokine expressed on mature dendritic cells (mDCs) has been investigated. We show that CX3CL1 promotes NK activation by mDCs. After blocking of CX3CL1 by antibody, no activation occurred but major histocompatibility complex (MHC) class I neutralization restored DC-mediated NK activation, suggesting an interaction between CX3CL1 signaling and the functioning of inhibitory KIR. Then the YTS NK cell line, in which the inhibitory receptor KIR2DL1 had been introduced, was used. The presence of KIR2DL1 did not decrease YTS activation by HLA-Cw4 DC when CX3CL1 was functional. In contrast, CX3CL1 neutralization led to killer cell immunoglobulin-like receptor (KIR) phosphorylation and SHP-1 recruitment in YTS(KIR2DL1) cultured with HLA-Cw4 mDCs. Moreover, CX3CL1 neutralization promoted dispersion of lipid rafts and the formation of a multiprotein complex required for cytoskeletal rearrangements in YTS NK cells. These findings point to a pivotal role of CX3CL1 in the activation of resting NK cells by mature DCs.     

Peripheral S-phase T cells in HIV disease have a central memory phenotype and rarely have evidence of recent T cell receptor engagement

Heightened proliferation and death of T lymphocytes may play a key role in human immunodeficiency virus (HIV) pathogenesis; however, the mechanism that mediates this effect and the phenotype of the proliferating T cells have not been clearly determined. We assessed S-phase cell frequencies and phenotype by ex vivo bromodeoxyuridine incorporation and flow-cytometric analysis in a group of 35 HIV-infected individuals. Frequencies of S-phase T cells were increased in HIV disease and were related to plasma HIV RNA levels but not to CD4 cell, total T cell, or total lymphocyte counts. S-phase cells were phenotypically defined as "central memory" cells (CD45RO+CD62L+CCR7+). Although activated (CD38+), S-phase cells lacked CD69 expression, rarely expressed CD25, and were not overrepresented among HIV-specific cells, as might have been expected if these cells had recently been activated by HIV antigens. Thus, in HIV infection, central memory T cells may be highly susceptible to bystander mechanisms of immune activation, leading to S-phase entry.     

Dendritic cells and HIV-specific CD4+ T cells: HIV antigen presentation, T-cell activation, and viral transfer

Human immunodeficiency virus (HIV)-specific CD4+ lymphocytes are preferentially infected in HIV-positive individuals. To study this preferential infection, we have derived several HIV-specific (HS) CD4+ clones. We show that in dendritic cells (DCs), HIV virion capture led to major histocompatibility complex class-II (MHC-II)-restricted viral antigen presentation and to activation of HS cells. In contrast, neither cell-free virions nor infected lymphocytes activated HS cells. In DCs, the dendritic cell-specific intercellular adhesion molecule 3-grabbing nonintegrin (DC-SIGN/CD209), which internalizes virions, promoted MHC-II presentation of HIV antigens. Activation of HS cells by HIV-exposed DCs triggered an efficient viral spread in lymphocytes. CD4+ clones with irrelevant antigenic specificities were not activated by HIV-exposed DCs and poorly supported viral replication under this setting. Our results unravel the mechanisms of MHC-II-restricted HIV antigen presentation by DCs and describe how HIV gains access to the very cells designed by the immune system to counteract this pathogen.     

Lytic versus stimulatory synapse in cytotoxic T lymphocyte/target cell interaction: manifestation of a dual activation threshold

Activation of biological functions in T lymphocytes is determined by the molecular dynamics occurring at the T cell/opposing cell interface. In the present study, a central question of cytotoxic T lymphocyte (CTL) biology was studied at the single-cell level: can two distinct activation thresholds for cytotoxicity and cytokine production be explained by intercellular molecular dynamics between CTLs and targets? In this study, we combine morphological approaches with numerical analysis, which allows us to associate specific patterns of calcium mobilization with different biological responses. We show that CTLs selectively activated to cytotoxicity lack a mature immunological synapse while exhibiting a low threshold polarized secretion of lytic granules and spike-like patterns of calcium mobilization. This finding is contrasted by fully activated CTLs, which exhibit a mature immunological synapse and smooth and sustained calcium mobilization. Our results indicate that intercellular molecular dynamics and signaling characteristics allow the definition of two activation thresholds in individual CTLs: one for polarized granule secretion (lytic synapse formation) and the other for cytokine production (stimulatory synapse formation).     

Chronic lymphocytic leukaemia: the role of T cells in a B cell disease

Chronic lymphocytic leukaemia (CLL) has long been thought to be an immunosuppressive disease and abnormalities in T-cell subset distribution and function have been observed in many studies. However, the role of T cells (if any) in disease progression remains unclear and has not been directly studied. This has changed with the advent of new therapies, such as chimeric antigen receptor-T cells, which actively use retargeted patient-derived T cells as “living drugs” for CLL. However complete responses are relatively low (~26%) and recent studies have suggested the differentiation status of patient T cells before therapy may influence efficacy. Non-chemotherapeutic drugs, such as idelalisib and ibrutinib, also have an impact on T cell populations in CLL patients. This review will highlight what is known about T cells in CLL during disease progression and after treatment, and discuss the prospects of using T cells as predictive biomarkers for immune status and response to therapy.     

Mast cells enhance T cell activation: Importance of mast cell-derived TNF

Mast cells are not only important effector cells in immediate hypersensitivity reactions and immune responses to pathogens but also can contribute to T cell-mediated disorders. However, the mechanisms by which mast cells might influence T cells in such settings are not fully understood. We find that mast cells can enhance proliferation and cytokine production in multiple T cell subsets. Mast cell-dependent enhancement of T cell activation can be promoted by FcepsilonRI-dependent mast cell activation, TNF production by both mast cells and T cells, and mast cell-T cell contact. However, at high concentrations of cells, mast cells can promote T cell activation independent of IgE or TNF. Finally, mast cells also can promote T cell activation by means of soluble factors. These findings identify multiple mechanisms by which mast cells can influence T cell proliferation and cytokine production.     

Dendritic cells acquire the MAGE-3 human tumor antigen from apoptotic cells and induce a class I-restricted T cell response

In an attempt to transduce monocyte-derived dendritic cells (DCs) with a retroviral vector coding for an intracytoplasmic tumor antigen (TAA), we were confronted by the evident dissociation between the ability of the treated DCs to induce a TAA-specific response, and the presence of integrated vector proviral DNA. The TAA, i.e., MAGE-3, was acquired by DCs and presented to immune effectors, thanks to the property of DCs to uptake the apoptotic bodies released by the irradiated vector-producing cells. Indeed, we observed that upon irradiation vector-producing cells underwent apoptotic cell death, monitored by annexin V and propidium iodide staining, and were phagocytosed by DCs. Lymphocytes obtained from a patient affected by a MAGE-3(+) melanoma, were stimulated in vitro with autologous DCs previously exposed to irradiated MAGE-3-expressing cells. This procedure led to the induction of MAGE-3-specific cytotoxic effectors, directed against a yet unknown MAGE-3 epitope presented by HLA-A*B5201 molecules. These data demonstrate that DCs can present engulfed human TAAs, thus providing strategies for cancer vaccination.     

Discordant responses during antiretroviral therapy: role of immune activation and T cell redistribution rather than true CD4 T cell loss

We studied T cell dynamics in four patients who initially responded well to highly active antiretroviral therapy (HAART) but subsequently experienced virological failure. Maintenance of peripheral blood CD4T cell counts was associated with low levels of immune activation. Low reactivity to rebounding virus may preserve normal T lymphocyte distribution over blood and tissues and be associated with stable peripheral blood T cell numbers in virological failures to HAART.     

CD4(+) T cell responses to cytomegalovirus in early life: a prospective birth cohort study

We compared cytomegalovirus (CMV)-specific interferon-gamma (IFN-gamma), interleukin 2 (IL-2), and CD154 CD4(+) T cell responses of infants to those from chronically infected adults and from children aged 4-5 years. Magnitudes of the responses were similar, although coexpression of IFN-gamma plus CD154 occurred more than coexpression of IFN-gamma plus IL-2 or IL-2 plus CD154. Responses remained constant during infancy, although the proportion of IFN-gamma-producing cells increased from infancy to adulthood. Most responding cells in infants were undifferentiated (i.e., CD27(+)CD28(+)), although IFN-gamma-producing cells were disproportionately CD27(-). By 12 months after diagnosis, viremia was rarely detectable, indicating that CMV was controlled despite the slow development of CMV-specific CD4(+) T cell responses.     

宫内感染乙型肝炎病毒免疫失败婴幼儿树突状细胞和T细胞亚群的变化

目的通过检测婴幼儿外周血树突状细胞和T细胞亚群的数量变化,探讨宫内感染乙型肝炎病毒导致免疫失败的机制。方法采用流式细胞仪技术和三色荧光染色法,对20例宫内感染免疫失败婴幼儿及29例宫内感染免疫成功婴幼儿进行外周血髓源性树突状细胞（MDC）、浆细胞样树突状细胞（PDC）和T细胞亚群检测。结果免疫成功组外周血MDC的百分比为（0.30±0.12）%,绝对数为（20.28±6.82）×106/L;PDC百分比为（0.28±0.12）%,绝对数为（18.01±5.58）×106/L;CD4＋百分比（39.55±8.80）%,CD8＋百分比（24.62±7.23）%,CD4＋/CD8＋为1.66±0.51。免疫失败组外周血MDC的百分比为（0.18±0.08）%,绝对数为（12.94±5.97）×106/L;PDC百分比为（0.16±0.09）%,绝对数为（11.75±7.36）×106/L;CD4＋百分比（32.94±7.58）%,CD8＋百分比（29.61±7.08）%,CD4＋/CD8＋为1.13±0.50。免疫失败组婴幼儿外周血2种DC的百分比、绝对数、CD4＋百分比、CD8＋百分比、CD4＋/CD8＋与免疫成...     

Protective immunity to cytomegalovirus (CMV) retinitis in AIDS is associated with CMV-specific T cells that express interferon- gamma and interleukin-2 and have a CD8+ cell early maturational phenotype

To determine potential correlates of immune recovery from AIDS-related cytomegalovirus retinitis (CMVR), multiparameter flow cytometry was used to characterize CMV-specific T cells from subjects with CMVR. Individuals with active retinitis were compared with those who had been clinically immunorestored by antiretroviral therapy and had > or =2 years of ophthalmologic follow-up without anti-CMV therapy or retinitis reactivation or progression. In comparison with patients with active retinitis, immunorestored patients had higher circulating CD4(+) and CD8(+) T cells expressing interleukin-2 and interferon- gamma in response to combined CMV pp65 and IE1 peptide pool stimulation. CD4(+) T cell responses were predominantly to pp65, whereas CD8(+) T cell responses were predominantly to IE. Immunorestored patients, compared with patients with active retinitis, had increased levels of circulating CMV-specific CD8(+) T cells with "early" (CD27(+)CD28(+)CD45RA(+), CD27(+)CD28(+)CD45RA(-)) and "intermediate" (CD27(-)CD28(+)CD45RA(-)) phenotypes. Recovery from AIDS-related CMVR after the initiation of antiretroviral therapy may be mediated by CMV-specific CD4(+) and CD8(+) T cells capable of promoting antigen-specific CD8(+) T cell proliferation.     

Dendritic cell targeted HIV gag protein vaccine provides help to a DNA vaccine including mobilization of protective CD8+ T cells

To improve the efficacy of T cell–based vaccination, we pursued the principle that CD4⁺ T cells provide help for functional CD8⁺ T cell immunity. To do so, we administered HIV gag to mice successively as protein and DNA vaccines. To achieve strong CD4⁺ T cell immunity, the protein vaccine was targeted selectively to DEC-205, a receptor for antigen presentation on dendritic cells. This targeting helped CD8⁺ T cell immunity develop to a subsequent DNA vaccine and improved protection to intranasal challenge with recombinant vaccinia gag virus, including more rapid accumulation of CD8⁺ T cells in the lung. The helper effect of dendritic cell-targeted protein vaccine was mimicked by immunization with specific MHC II binding HIV gag peptides but not peptides from a disparate Yersinia pestis microbe. CD4⁺ helper cells upon adoptive transfer allowed wild-type, but not CD40^−/−, recipient mice to respond better to the DNA vaccine. The transfer also enabled recipients to more rapidly accumulate gag-specific CD8⁺ T cells in the lung following challenge with vaccinia gag virus. Thus, complementary prime boost vaccination, in which prime and boost favor distinct types of T cell immunity, improves plasmid DNA immunization, including mobilization of CD8⁺ T cells to sites of infection.     

Induction of costimulation of human CD4 T cells by tumor necrosis factor-related apoptosis-inducing ligand: possible role in T cell activation in systemic lupus erythematosus

OBJECTIVE: Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) has recently been shown to induce costimulation of mouse T cells in conjunction with signals from the T cell receptor. This study was undertaken to investigate TRAIL-induced costimulation of human T cells in order to determine the role of TRAIL-induced T cell activation in human systemic lupus erythematosus (SLE). METHODS: An in vitro T cell stimulation system with immobilized anti-CD3 and recombinant TRAIL receptor DR4-Fc proteins was used to activate human T cells purified from healthy individuals and from patients with SLE. The T cells were stimulated in vitro to assay their proliferation response by (3)H-thymidine incorporation, and their cytokine production by enzyme-linked immunosorbent assay. Activation of p38 MAPK after TRAIL stimulation was detected with specific anti-phospho-p38 MAPK monoclonal antibodies in Western blots. RESULTS: Enhanced T cell proliferation and increased interleukin-2 and interferon-gamma (IFNgamma) production were demonstrated in human T cells after stimulation with immobilized DR4-Fc and anti-CD3 in vitro. TRAIL engagement selectively activated human CD4, rather than CD8, T cells and augmented IFNgamma production. Activation of p38 MAPK was detected after TRAIL-induced T cell activation. T cells isolated from patients with SLE demonstrated a stronger response to TRAIL-induced costimulation, in terms of proliferation and increased up-regulation of CD25 after activation, when compared with T cells from healthy subjects. CONCLUSION: TRAIL engagement induces costimulation of human CD4 T cells via a p38 MAPK-dependent pathway. The results suggest that enhanced reactivity of T cells to autoantigens as a result of TRAIL-induced costimulation may play a role in the development of human autoimmune diseases.     

Analysis of the linker for activation of T cells and the linker for activation of B cells in natural killer cells reveals a novel signaling cassette, dual usage in ITAM signaling, and influence on development of the Ly49 repertoire

The linker for activation of T cells (LAT) and the linker for activation of B cells (LAB/NTAL/LAT2) are integral proteins in receptor coupling to downstream events. Both proteins are expressed in natural killer (NK) cells and LAT is phosphorylated during target cell interactions or ligation of the immunoreceptor tyrosine-based activation motif (ITAM)-coupled CD16. Regardless, Lat(-/-) mice exhibit normal natural and antibody-mediated killing. Here we place both LAT and LAB in the DAP12 pathway of NK cells. Moreover, we unveil a LAT-independent pathway that requires expression of Syk. Mice lacking either LAT or LAB have a skewed Ly49 repertoire, and activated NK cells from Lat(-/-) mice have reduced responses to the ITAM-coupled receptor NK1.1. In contrast, resting Lat(-/-) NK cells show intact NK1.1 responses, whereas NK cells without LAB are hyperactive. Elimination of both adaptors severely reduces NK1.1 signaling under both conditions. Together these data show that NK ITAMs preferentially use a signaling cassette regulated by interplay between LAT and LAB. Activation by interleukin-2 causes a shift to greater dependency on LAT due to suppression of Syk signaling. The overlapping use of multiple adaptors permits fine-tuning of NK-cell ITAM responses over the course of an immune response.