Indirect activation of naïve CD4+ T cells by dendritic cell-derived exosomes

Dendritic cells (DCs) secrete vesicles of endosomal origin, called exosomes, that bear major histocompatibility complex (MHC) and T cell costimulatory molecules. Here, we found that injection of antigen- or peptide-bearing exosomes induced antigen-specific na?ve CD4+ T cell activation in vivo. In vitro, exosomes did not induce antigen-dependent T cell stimulation unless mature CD8alpha- DCs were also present in the cultures. These mature DCs could be MHC class II-negative, but had to bear CD80 and CD86. Therefore, in addition to carrying antigen, exosomes promote the exchange of functional peptide-MHC complexes between DCs. Such a mechanism may increase the number of DCs bearing a particular peptide, thus amplifying the initiation of primary adaptive immune responses.     

High efficiency of reprogramming CD34⁺ cells derived from human amniotic fluid into induced pluripotent stem cells with Oct4

Although many techniques can be used to generate multitype-induced pluripotent stem (iPS) cells from multitype seed cells, improving the efficiency and shortening the period of cell reprogramming remain troublesome issues. In this study, to generate iPS cells, CD34? cells, isolated from human amniotic fluid cells (HuAFCs) by flow cytometry, were infected with retroviruses carrying only one reprogramming factor (Oct4) and cultured on human amniotic epithelial cell (HuAEC) feeder layers. Approximately 4 to 5 days after viral infection, some embryonic stem cell (ESC)-like colonies appeared among the feeder cells. These colonies were positive for alkaline phosphatase and expressed high levels of ESC pluripotent markers (Nanog, Sox2, Oct4, CD133, and Rex1). Moreover, these iPS cells exhibited high levels of telomerase activity and had normal karyotypes. Additionally, these cells could differentiate into cell types from all 3 germ layers in vivo and in teratomas. In summary, we report a novel way of iPS generation that uses CD34? HuAFCs as seed cells. Using this method, we can generate human iPS cells with greater efficiency and safety (the oncogenic factors, c-Myc and Klf4, were not used), and using the minimum number of reprogramming factors (only one factor, Oct4). Besides, HuAECs were used as feeder layers to culture human iPS cells, which could not only avoid contamination with heterogeneous proteins, but also maintain iPS cells in a self-renewing and undifferentiated state for a long time.     

CD103−CD11b+ dendritic cells regulate the sensitivity of CD4 T-cell responses to bacterial flagellin

Toll-like receptor 5 (TLR5) has been widely studied in an inflammatory context, but the effect of TLR5 on the adaptive response to bacterial flagellin has received considerably less attention. Here, we demonstrate that TLR5 expression by dendritic cells (DCs) allows a 1,000-fold enhancement of T-cell sensitivity to flagellin, and this enhancement did not require the expression of NLRC4 or Myd88. The effect of TLR5 on CD4 T-cell sensitivity was independent of the adjuvant effect of flagellin and TLR5 ligation did not alter the sensitivity of ovalbumin (OVA)-specific T cells to OVA. In the spleen, the exquisite T-cell sensitivity to flagellin was regulated by CD4−CD8α− DCs and was blocked by a monoclonal antibody to TLR5. In the mesenteric lymph nodes, flagellin-specific T-cell activation was regulated by a population of CD103−CD11b+ DCs. Thus, TLR5 expression by mucosal and systemic DC subsets controls the sensitivity of the adaptive immune response to flagellated pathogens.     

Lymph-borne CD8α+ dendritic cells are uniquely able to cross-prime CD8+ T cells with antigen acquired from intestinal epithelial cells

Cross-presentation of cellular antigens is crucial for priming CD8⁺ T cells, and generating immunity to intracellular pathogens—particularly viruses. It is unclear which intestinal phagocytes perform this function in vivo. To address this, we examined dendritic cells (DCs) from the intestinal lymph of IFABP-tOVA 232-4 mice, which express ovalbumin in small intestinal epithelial cells (IECs). Among lymph DCs (LDCs) only CD103⁺ CD11b⁻ CD8α⁺ DCs cross-present IEC-derived ovalbumin to CD8⁺ OT-I T cells. Similarly, in the mesenteric lymph nodes (MLNs), cross-presentation of IEC–ovalbumin was limited to the CD11c⁺ MHCII^hi CD8α⁺ migratory DCs, but absent from all other subsets, including the resident CD8α^hi DCs. Crucially, delivery of purified CD8α⁺ LDCs, but not other LDC subsets, into the MLN subcapsular lymphatic sinus induced proliferation of ovalbumin-specific, gut-tropic CD8⁺ T cells in vivo. Finally, in 232-4 mice treated with R848, CD8α⁺ LDCs were uniquely able to cross-prime interferon γ-producing CD8⁺ T cells and drive their migration to the intestine. Our results clearly demonstrate that migrating CD8α⁺ intestinal DCs are indispensable for cross-presentation of cellular antigens and, in conditions of inflammation, for the initial differentiation of effector CD8⁺ T cells. They may therefore represent an important target for the development of antiviral vaccinations.     

Immune tolerance induced by intravenous transfer of immature dendritic cells via up-regulating numbers of suppressive IL-10+ IFN-γ+-producing CD4+ T cells

Dendritic cells (DCs) regulate immunity and immune tolerance in vivo. However, the mechanisms of DC-mediated tolerance have not been fully elucidated. Here, we demonstrate that intravenous (i.v.) transfer of bone marrow-derived DCs pulsed with myelin oligodendrocyte glycoprotein (MOG) peptide blocks the development of experimental autoimmune encephalomyelitis in C57BL/6J mice. i.v. transfer of MOG-pulsed DCs leads to the down-regulation of the production of IL-17A and IFN-γ and up-regulation of IL-10 secretion. The development of regulatory T cells (Tregs) is facilitated via up-regulation of FoxP3 expression and production of IL-10. The number of suppressive CD4⁺IL-10⁺IFN-γ⁺ T cells is also improved. The expression of OX40, CD154, and CD28 is down-regulated, but the expression of CD152, CD80, PD-1, ICOS, and BTLA is up-regulated on CD4⁺ T cells after i.v. transfer of immature DCs. The expression of CCR4, CCR5, and CCR7 on CD4⁺ T cells is also improved. Our results suggest that immature DCs may induce tolerance via facilitating the development of CD4⁺FoxP3⁺ Tregs and suppressive CD4⁺IL-10⁺IFN-γ⁺ T cells in vivo.     

Selective expansion of perforin-positive CD8+ T cells by immature dendritic cells infected with live Bacillus Calmette-Guérin mycobacteria

Tyrosine phosphorylation is thought to be critical in the regulation of neutrophil functioning, and members of the Src family of tyrosine kinases have recently been shown to be regulated in activated granulocytes. We have used a specific pharmacological inhibitor of Src kinases, pyrazolpyrimidine 1 (PP1), to evaluate the role of Src kinases in cytokine/chemoattractant-induced regulation of neutrophil function. PP1 inhibits PKB phosphorylation but not STAT5 phosphorylation or the activation of MAP kinases by fMLP or GM-CSF. Pretreatment of neutrophils with PP1 and with the PI3K inhibitor LY294002 resulted in a strong inhibition of fMLP-induced superoxide production and cytokine-mediated survival but not fMLP-induced migration. It is interesting that the kinetics of inhibition of actin polymerization and the respiratory burst are very similar. Although initiation of both processes was not affected, sustained activation was inhibited by PP1. Taken together, our results demonstrate a critical role for Src kinases in regulating neutrophil cytotoxic-effector functioning through PI3K-PKB.     

CCR2+CD103− intestinal dendritic cells develop from DC-committed precursors and induce interleukin-17 production by T cells

The identification of intestinal macrophages (mφs) and dendritic cells (DCs) is a matter of intense debate. Although CD103⁺ mononuclear phagocytes (MPs) appear to be genuine DCs, the nature and origins of CD103⁻ MPs remain controversial. We show here that intestinal CD103⁻CD11b⁺ MPs can be separated clearly into DCs and mφs based on phenotype, gene profile, and kinetics. CD64⁻CD103⁻CD11b⁺ MPs are classical DCs, being derived from Flt3 ligand-dependent, DC-committed precursors, not Ly6C^hi monocytes. Surprisingly, a significant proportion of these CD103⁻CD11b⁺ DCs express CCR2 and there is a selective decrease in CD103⁻CD11b⁺ DCs in mice lacking this chemokine receptor. CCR2⁺CD103⁻ DCs are present in both the murine and human intestine, drive interleukin (IL)-17a production by T cells in vitro, and show constitutive expression of IL-12/IL-23p40. These data highlight the heterogeneity of intestinal DCs and reveal a bona fide population of CCR2⁺ DCs that is involved in priming mucosal T helper type 17 (Th17) responses.     

TGF-β1 gene-modified,immature dendritic cells delay the development of inflammatory bowel disease by inducing CD4+Foxp3+ regulatory T cells

Inflammatory bowel disease (IBD) is caused by an uncontrolled immune response in the intestinal lumen, leading to inflammation in genetically predisposed individuals. Immunotherapy may be a promising approach to the treatment of IBD. Here, we show that transforming growth factor-β1 (TGF-β1) gene-modified immature dendritic cells (imDCs) could enhance the inhibitory function of imDCs and delay the progress of IBD induced by dextran sodium sulfate in mice. The results of fluorescence-activated cell sorter (FACS) demonstrated that this protective effect is mediated partially by inducing CD4⁺Foxp3⁺ regulatory T cells (Tregs) in mesentery lymph nodes to control inflammation. In vitro experiments also supported this hypothesis. In conclusion, we provide evidence that TGF-β1-modified bone marrow-derived imDCs may have a therapeutic effect to IBD.     

Robust interferon-α and IL-12 responses by dendritic cells are related to efficient CD4+ T-cell recovery in HIV patients on ART

Amongst HIV patients with successful virological responses to antiretroviral therapy (ART), poor CD4(+) T-cell recovery is associated with low nadir CD4(+) T-cell counts and persistent immune activation. These factors might be influenced by dendritic cell (DC) function. Interferon-α-producing plasmacytoid DC and IL-12-producing myeloid DC were quantified by flow cytometry after stimulation with agonists to TLR7/8 (CL075) or TLR9 (CpG-ODN). These were compared between patients who achieved CD4(+) T-cell counts above or below 200 cells/μL after 6 months on ART (High vs. Low groups). High Group patients had more DC producing interferon-α or IL-12 at Weeks 6 and 12 on ART than Low Group patients. The frequencies of cytokine-producing DC at Week 12 were directly correlated with CD4(+) T-cell counts at baseline and at Week 12. Patients with good recovery of CD4(+) T-cells had robust TLR-mediated interferon-α responses by plasmacytoid DC and IL-12 responses by myeloid DC during early ART (1-3 months).     

TGF-β1 and contact mediated suppression by CD4+CD25+CD127− T regulatory cells of patients with self-limiting hepatitis E

Background and aimLiterature on the role of Regulatory T cells (Tregs) in acute viral infections is limited. Having established that the Tregs in self-limiting hepatitis E infection are elevated and functional, this study has focused on characterizing the specificity, phenotypes and identifying the molecules or factors responsible for enhancement of Treg cells and abrogation of Treg-mediated suppression in hepatitis E.MethodsHEV rORF2p specific (a) Treg frequency, subset analysis and expression of surface and intracellular markers on Tregs and CFSE based functional analysis by flow cytometry (b) key cytokines quantification by multiplex (c) suppressive functional assay in the presence of anti-TGF-β₁ or anti-IL-10 or both antibodies or Transwell insert or in combination were performed on samples from 58 acute patients (AVH-E), 45 recovered individuals from hepatitis E and 55 controls.ResultsIn AVH-E, the increased frequencies of Tregs and Teff cells were HEV rORF2p specific and Treg cells were of effector memory phenotype. Higher expressions of HEV rORF2p stimulated CTLA-4, GITR, PD1L, CD103, CD39, TLR2 and TGF-β₁ molecules on Tregs of AVH-E were observed. Tregs produced TGF-β₁ and inhibited the secretion of IFN-γ. Transwell insert and cytokines blocking assays indicated Tregs mediated suppression in AVH-E patients is majorly TGF-β₁ mediated and partly cell-cell contact mediated.ConclusionOverall, we have identified beneficial involvement of HEV specific, functional Tregs and TGF-β₁ as the regulatory molecule responsible for enhancement of Tregs in self-limiting HEV infection. Therefore, use of TGF-β₁ as a possible supplement for boosting Treg response in recovery from severe hepatitis E needs evaluation.     

The stimulation of CD8+ T cells by dendritic cells pulsed with polyketal microparticles containing ion-paired protein antigen and poly(inosinic acid)–poly(cytidylic acid)

New adjuvants and delivery strategies are needed to optimize the ability of protein-based vaccines to elicit CD8⁺ T cell responses. We have developed a model vaccine formulation containing ovalbumin (OVA) and the double-stranded RNA analog poly(inosinic acid)–poly(cytidylic acid) (poly(I:C)), a TLR3 agonist. OVA and poly(I:C) were each ion-paired to cetyltrimethylammonium bromide (CTAB) to produce hydrophobic complexes, which were co-encapsulated in pH-sensitive polyketal (PK3) microparticles (1–3 μm) using a single emulsion method. Loading levels ranged from 13.6 to 18.8 μg/mg OVA and 4.8 to 10.3 μg/mg poly(I:C). Murine splenic dendritic cells (DCs) pulsed with PK3-OVA–poly(I:C) microparticles, at antigen doses of 0.01 and 0.1 μg/mL, induced a higher percentage of IFNγ-producing CD8⁺ T cells than DCs treated with PK3-OVA particles or soluble OVA/poly(I:C). A higher antigen dose (1 μg/mL) was less effective, which can be attributed to CTAB toxicity. At the lowest antigen dose (0.01 μg/mL), PK3-OVA–poly(I:C) microparticles also enhanced TNF-α and IL-2 production in CD8⁺ T cells. These data demonstrate the potential of polyketal microparticles in formulating effective CD8⁺ T cell-inducing vaccines comprising protein antigens and dsRNA adjuvants.     

Enhanced protection conferred by mucosal BCG vaccination associates with presence of antigen-specific lung tissue-resident PD-1+ KLRG1− CD4+ T cells

BCG, the only vaccine licensed against tuberculosis, demonstrates variable efficacy in humans. Recent preclinical studies highlight the potential for mucosal BCG vaccination to improve protection. Lung tissue-resident memory T cells reside within the parenchyma, potentially playing an important role in protective immunity to tuberculosis. We hypothesised that mucosal BCG vaccination may enhance generation of lung tissue-resident T cells, affording improved protection against Mycobacterium tuberculosis. In a mouse model, mucosal intranasal (IN) BCG vaccination conferred superior protection in the lungs compared to the systemic intradermal (ID) route. Intravascular staining allowed discrimination of lung tissue-resident CD4⁺ T cells from those in the lung vasculature, revealing that mucosal vaccination resulted in an increased frequency of antigen-specific tissue-resident CD4⁺ T cells compared to systemic vaccination. Tissue-resident CD4⁺ T cells induced by mucosal BCG displayed enhanced proliferative capacity compared to lung vascular and splenic CD4⁺ T cells. Only mucosal BCG induced antigen-specific tissue-resident T cells expressing a PD-1⁺ KLRG1⁻ cell-surface phenotype. These cells constitute a BCG-induced population which may be responsible for the enhanced protection observed with IN vaccination. We demonstrate that mucosal BCG vaccination significantly improves protection over systemic BCG and this correlates with a novel population of BCG-induced lung tissue-resident CD4⁺ T cells.     

Targeting Janus tyrosine kinase 3 (JAK3) with an inhibitor induces secretion of TGF-β by CD4+ T cells

Regulatory T cells (Tregs) are critical for the peripheral maintenance of the autoreactive T cells in autoimmune disorders such as type 1 diabetes (T1D). Pharmacological inhibition of Janus tyrosine kinase 3 (JAK3) has been proposed as a basis for new treatment modalities against autoimmunity and allogeneic responses. Targeting JAK3 with an inhibitor has previously been shown to exhibit protective action against the development of T1D in non-obese diabetic (NOD) mice. As the mechanism of such preventative action has been unknown, we hypothesized that JAK3 inhibition induces generation of Tregs. Here, we show that the JAK3 inhibitor 4-(4′-hydroxyphenyl)-amino-6,7-dimethoxyquinazoline (WHI-P131) suppresses proliferation of short-term cultured NOD CD4⁺ T cells through induction of apoptosis, while promoting survival of a particular population of long-term cultured cells. It was found that the surviving cells were not of the CD4⁺CD25⁺FoxP3⁺ phenotype. They secreted decreased amounts of IL-10, IL-4 and interferon (IFN)-γ compared to the cells not exposed to the optimal concentrations of JAK3 inhibitor. However, an elevated transforming growth factor (TGF)-β secretion was detected in their supernatants. In vivo treatment of prediabetic NOD mice with WHI-P131 did not affect the frequency and number of splenic and pancreatic lymph node CD4⁺FoxP3⁺ Tregs, while generating an elevated numbers of CD4⁺FoxP3⁻ TGF-β-secreting T cells. In conclusion, our data suggest an induction of TGF-β-secreting CD4⁺ T cells as the underlying mechanism for antidiabetogenic effects obtained by the treatment with a JAK3 inhibitor. To our knowledge, this is the first report of the JAK3 inhibitor activity in the context of the murine Tregs.