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The characteristics and functions of CD4 + CD25 + regulatory T cells (Tregs) have been well defined in murine and human systems. However, the interaction or crosstalk between CD4 + CD25 + Tregs and dendritic cells (DCs) remains controversial. In this study, the effects of chronic hepatitis B (CHB) CD4 + CD25 + Tregs on the maturation and function of monocyte-derived DCs were examined. The results showed that CD4 + CD25 + render the DCs inefficient as antigen-presenting cells (APCs) despite prestimulation with CD40 ligand. This effect was marginally reverted by applying neutralizing antibodies (Abs) to IL-10 and TGF-β. There were an increased IL-10 and TGF-β secretion and reduced expression of costimulatory molecules in DC. Thus, in addition to a direct suppressor effect on CD4 + T cells, CD4 + CD25 + may modulate the immune response through DCs in CHB patients., (© 2018 John Wiley & Sons Ltd.)

Aim: Porphyromonas gingivalis can synthesize an extracellular capsule and different serotypes have been described based on capsular antigenicity. On dendritic cells (DCs), the type of capsule present plays a role on the strength of the developed immune response. This study aimed to investigate the T-lymphocyte responses when stimulated with autologous mature DCs exposed to different P. gingivalis K-serotypes., Materials and Methods: Naïve CD4(+) T-lymphocytes were obtained from healthy subjects and stimulated with autologous DCs primed with increasing multiplicity of infections of the different P. gingivalis K-serotypes. The Th1, Th2, Th17 and T-regulatory cytokines and transcription factor levels were quantified., Results: Distinct types of response were detected when T-lymphocytes were stimulated by DCs primed with the different P. gingivalis K-serotypes. T-lymphocytes stimulated by K1 or K2-primed DCs elicited higher levels of Th1 and Th17-associated cytokines, T-bet and RORC2 than T-lymphocytes stimulated with DCs primed with the other serotypes. Conversely, the serotypes K3-K5 induced higher levels of Th2-associated cytokines and GATA-3 than the others., Conclusions: These results demonstrate that DCs primed with the different P. gingivalis K-serotypes elicited distinct T-cell responses. Strains K1 (W83) and K2 (HG184) induced a Th1/Th17 pattern of immune response and K3 (A7A1-28), K4 (ATCC(®49417™) ), and K5 (HG1690) a Th2 response., (© 2013 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Cancer immunotherapeutic agents (vaccines) in the form of antigen-loaded dendritic cells (DCs) reached an important milestone with the recent approval of Provenge, the first DC vaccine for treatment of prostate cancer. Although this heralds a new era of tumor immunotherapy, it also highlights the compelling need to optimize such DC-based therapies as they are increasingly tested and used to treat human patients. In this study we sought to augment and enhance the antitumor activity of a DC-based vaccine using siRNA to silence expression of immunosuppressive enzyme indoleamine 2,3-dioxygenase (IDO) in DCs. We report here that DCs loaded with tumor antigens, but with siRNA-silenced IDO expression, were introduced into 4T1 breast tumor-bearing mice, the treatment: (i) lengthened the time required for tumor onset, (ii) decreased tumor size compared to tumors grown for equal lengths of time in mice treated with antigen-loaded DCs without IDO silencing and (iii) reduced CD4(+) and CD8(+) T cell apoptosis. Furthermore, immunization with IDO-silenced DCs enhanced tumor antigen-specific T cell proliferation and CTL activity, and decreased numbers of CD4(+) CD25(+) Foxp3(+) T(reg). This study provides evidence to support silencing of immunosuppressive genes (IDO) as an effective strategy to enhance the efficacy of DC-based cancer immunotherapeutic., (Copyright © 2012 UICC.)

Along with their immunogenic role, dendritic cells (DCs) are also critical in maintaining tolerance to self-antigens by inducing regulatory T cells (Tregs) via the expression of the immunomodulatory enzyme indoleamine 2,3-dioxygenase 1 (IDO1). In turn, Tregs modulate the maturation and/or function of DCs. In immune thrombocytopenia (ITP), the interaction between DCs and Tregs has never been investigated although decreased number/function of Tregs as well as altered DCs have been described. Here, we ask whether, in ITP: (1) IDO1 expression/activity is decreased in mature DCs; (2) IDO1-mediated Treg generation is impaired; and (3) DC maturation is abnormally modulated by Tregs. We found that in ITP, DCs show reduced capability of upregulating the expression/activity of IDO1. This finding results in the reduced ability of mature DCs of converting T cells into Tregs. In turn, Tregs are characterized by decreased interleukin-10 production and show lower ability of inhibiting DC maturation. In conclusion, these data point out the role of IDO1 in the impaired regulatory T cell development of ITP patients and suggest that the cross-talk between Tregs and DCs is hampered and plays a pathogenetic role.

Background: Alveolar echinococcosis, caused by Echinococcus multilocularis larvae, is a chronic disease associated with considerable modulation of the host immune response. Dendritic cells (DC) are key effectors in shaping the immune response and among the first cells encountered by the parasite during an infection. Although it is assumed that E.multilocularis, by excretory/secretory (E/S)-products, specifically affects DC to deviate immune responses, little information is available on the molecular nature of respective E/S-products and their mode of action., Methodology/principal Findings: We established cultivation systems for exposing DC to live material from early (oncosphere), chronic (metacestode) and late (protoscolex) infectious stages. When co-incubated with Echinococcus primary cells, representing the invading oncosphere, or metacestode vesicles, a significant proportion of DC underwent apoptosis and the surviving DC failed to mature. In contrast, DC exposed to protoscoleces upregulated maturation markers and did not undergo apoptosis. After pre-incubation with primary cells and metacestode vesicles, DC showed a strongly impaired ability to be activated by the TLR ligand LPS, which was not observed in DC pre-treated with protoscolex E/S-products. While none of the larvae induced the secretion of pro-inflammatory IL-12p70, the production of immunosuppressive IL-10 was elevated in response to primary cell E/S-products. Finally, upon incubation with DC and naïve T-cells, E/S-products from metacestode vesicles led to a significant expansion of Foxp3+ T cells in vitro., Conclusions: This is the first report on the induction of apoptosis in DC by cestode E/S-products. Our data indicate that the early infective stage of E. multilocularis is a strong inducer of tolerance in DC, which is most probably important for generating an immunosuppressive environment at an infection phase in which the parasite is highly vulnerable to host attacks. The induction of CD4+CD25+Foxp3+ T cells through metacestode E/S-products suggests that these cells fulfill an important role for parasite persistence during chronic echinococcosis.

Our and others' previous studies have shown that Schistosoma japonicum (SJ) infection can inhibit allergic reactions. Moreover, we found that adoptive transfer of dendritic cells (DCs) from inhibited mice showed a similar inhibitory effect on allergy, suggesting a critical role of DCs in SJ-infected mediated inhibition of allergy. In this study, we further examined the mechanism by which DCs contribute to inhibition of allergy. Our results showed that DCs from SJ-infected mice (SJDCs) produced significantly higher levels of IL-10 compared to those from naive control mice (NDCs). Adoptive transfer of SJDCs, unlike NDCs, significantly increased CD4+CD25+Foxp3+ T cells and CD4+CD25+IL-10+ T cells regulatory T-cell responses in vivo. This was correlated with significantly reduced production of IL-4 and IL-5 by CD4+ T cells, eotaxin in lung tissues and reduced airway allergic inflammation in the SJDC recipients following allergen sensitization and challenge. These data suggest that helminth infection may induce tolerogenic DCs that can inhibit the development of airway allergic inflammation through enhancing T regulatory cell responses., (© 2011 Blackwell Publishing Ltd.)

We compared the effects of yeast-treated human dendritic cells (DCs) with CD40L-matured human DCs for the induction of effector cells and the number and functionality of CD4(+)CD25(+)CD127(-)FoxP3(+) regulatory T cells (Tregs). DCs were treated with yeast or CD40L and cocultured with isolated autologous CD4(+) T cells. CD4(+)CD25(+)CD127(-) T cells isolated from the coculture of CD4(+) T cells plus yeast-treated DCs (yeast coculture) had a lower expression of FoxP3 and decreased suppressive function compared to CD4(+)CD25(+)CD127(-) T cells isolated from the coculture of CD4(+) T cells plus CD40L-treated DCs (CD40L coculture). Also, compared to the CD40L coculture, the yeast coculture showed increases in the ratio of CD4(+)CD25(+) activated T cells to Tregs and in the production of Th1-related cytokines (IL-2, TNF-α, IFN-γ) and IL-6. In addition, yeast-treated DCs used as antigen-presenting cells (APCs) incubated with the tumor antigen CEA enhanced the proliferation of CEA-specific CD4(+) T cells compared to the use of CD40L-matured DCs used as APCs. This is the first study to report on the role of yeast-treated/matured human DCs in reducing Treg frequency and functionality and in enhancing effector to Treg ratios. These results provide an additional rationale for the use of yeast as a vector in cancer vaccines., (Published by Elsevier Ltd.)

Dendritic cells (DCs) are specialized antigen-presenting cells (APCs) required for T-cell activation and are classified into several subtypes by phenotypic and functional characteristics. However, it remains unclear if distinct transcription factors control the development of each DC subpopulation. In this report, we demonstrate that Notch signaling controls the development of a novel DC subtype that expresses Thy1 (Thy1(+) DCs). Overstimulation of bone marrow cells with the Notch ligand Delta-like 1 promoted the development of Thy1(+) DCs. Thy1(+) DCs are characterized as CD11c(+) MHC class II(+) NK1.1(-) B220(-) CD8α(+) , and are present in the thymus but not in the spleen and lymph nodes. Thymic Thy1(+) DCs are able to capture exogenous proteins and delete CD4(+) CD8(+) T cells. Transplantation experiments demonstrated that CD44(+) CD25(-) and CD44(+) CD25(+) thymocytes can differentiate into Thy1(+) DCs. Recombination signal binding protein for immunoglobulin kappa J region (RBP-J) deficiency in lineage-negative bone marrow cells, but not CD11c(+) cells, disrupted Thy1(+) DC development in the thymus. Our data indicate that Notch signaling controls the development of a novel type of Thy1-expressing DC in the thymus that possibly controls negative selection, and indicates that there may be highly regulated, differential transcriptional control of DC development. Furthermore, our findings suggest that Notch signaling regulates T-cell development not only by intrinsically inducing T-cell lineage-specific gene programs, but also by regulating negative selection through Thy1(+) DCs., (Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

CD4+CD25+ regulatory T cells (Tregs) are known to inhibit immune responses to antigens. Since, the process of antigen uptake by dendritic cells (DC) is central to induction of immune responses, we analyzed the effect of Tregs on the expression of endocytic receptors on DC and its repercussion on antigen uptake. Our results demonstrate that Tregs down-regulate the expression and uptake of antigens via C-type lectin-like receptors CD206 and DC-SIGN, restrain the pinocytosis process of DC and augment the expression of FcγRIIB, an inhibitory Fcγ receptor the engagement of which by IgG-bound antigens leads to inhibition of DC activation. Our results thus provide an additional insight on the pertinence of strategies aimed at blocking Treg functions towards improved vaccination protocols., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Objectives: Researchers recently discovered a group of semimature dendritic cells that induce autoimmune tolerance by activating host antigen-specific CD4+CD25+ T-regulatory cells. We hypothesized that donor semimature dendritic cells injected into recipients would induce effector T-cell hyporesponsiveness by activating CD4+CD25+ T-regulatory cells., Materials and Methods: Donor myeloid semimature dendritic cells were cultivated for 6 days and were then stimulated with tumor necrosis factor a for 24 hours. BALB/c mice were pretreated with semimature dendritic cells to generate antigen-specific CD4+CD25+ T-regulatory cells in vivo. The role of CD4+CD25+ T-regulatory cells in transplant immunity was studied via mixed lymphocyte culture in vitro., Results: Surface markers and cytokines secreted by semimature dendritic cells differed from those secreted by immature myeloid dendritic cells or mature dendritic cells. Semimature dendritic cells and immature myeloid dendritic cells did not activate allogenic lymphocyte responses in coculture studies. CD4+CD25+ T-regulatory cells of recipients challenged by donor semimature dendritic cells, which expressed a high level of interleukin-10, induced hyporesponsiveness in host effector T cells that were stimulated by donor splenocytes. In contrast, CD4+CD25+ T-regulatory cells did not induce hyporesponsiveness in effector T cells when the host T cells were stimulated by third-party antigen from DBA2 mice splenocytes., Conclusions: Our findings confirm that semimature dendritic cells are an independent subgroup of dendritic cells in both immune function and morphologic profile. It may be the cytokine secretion profile of semimature dendritic cells (rather than that of surface markers) that has a key role in inducing CD4+CD25+ T-regulatory cells to express a high level of interleukin-10. Immunization with donor semimature dendritic cells may be an effective method of inducing transplant tolerance, but further evidencebased studies of that topic are necessary.

Background & Aims: Orally induced tolerance to environmental allergens prevents deleterious, systemic, delayed-type hypersensitivity responses via immune suppression mechanisms believed to include either anergy/deletion of specific effector T cells or active suppression by CD4(+)CD25(+) regulatory T cells (Tregs). The aim of this study was to investigate whether and how antigen (Ag) penetration through the gut orchestrates these 2 distinct mechanisms., Methods: Using a model of allergic contact dermatitis (ACD) mediated by hapten-specific cytolytic CD8(+) T cells and a T-cell transfer model of contact hypersensitivity in CD3epsilon-deficient mice, we studied the outcome of Ag gavage on CD8(+) effectors and Tregs., Results: Full protection from ACD by gavage with the relevant allergen required 2 coordinated events taking place first in gut-associated lymphoid tissues and then systemically. Allergen gavage induced deletion by plasmacytoid dendritic cells of a large fraction of Ag-specific CD8(+) T cells in liver and mesenteric lymph nodes and also triggered the suppressive function of Treg of secondary lymphoid organs. Residual Ag-specific CD8(+) T cells conditioned during this mucosal step are fully susceptible to suppression by activated Treg, which completely prevented their differentiation into ACD effectors, upon re-exposure to the allergen via the skin., Conclusions: Thus, oral tolerance initiated in gut-associated lymphoid tissues by the plasmacytoid dendritic cells-mediated deletion of Ag-specific T cells is completed systemically by CD4(+)CD25(+) T cells. Biotherapies able to increase the susceptibility of effector T cells to the suppressive function of Treg may be valuable for the treatment of autoimmune and inflammatory diseases.

Dendritic cells (DCs) play a pivotal role in linking the innate immunity and acquired immunity in responses to pathogen. Non-human primates such as Chinese Rhesus Macaque (CRM) are the favorable models for preclinical study of potential therapeutic drugs, vaccines and mechanisms of human diseases. However, the phenotypical characterization of monocyte-derived dendritic cells (MDDCs) from CRM has not been elucidated. Monocytes from CRM were cultured with GM-CSF and IL-4 in RPMI-1640. Six days later, these cells were differentiated with typical dendritical morphology. CD11c and DC-SIGN were highly expressed. The immature MDDCs expressed the low levels of CD25, CD80, CD83, moderate CD40, CD86, and high MHC. After stimulation, the mature MDDCs increased expression of mature molecules CD25 and CD83, co-stimulatory molecules such as CD80, CD86 and CD40, and kept a high level of MHC. The capacity of endocytosis decreased with maturation. The mature MDDCs have strong ability of inducing allogeneic T cell proliferation and producing IL-12. In conclusion, we have characterized the phenotype and ultimate function of MDDCs from CRM for the first time.

Variety of positive as well as negative regulatory signals are provided by antigen presenting cell in particular by dendritic cells. In this research, we studied the capacity of dendritic cells to expand antigen-specific T regulatory cells.We also investigated the role of TGF-beta in induction inhibitory functions of dendritic cells in mixed leukocyte reactions.Dendritic cells were generated from blood CD14(+) monocytes with granulocyte-Monocyte colony stimulating factor and interleukin-4 with or without TGF-beta (TGF-beta-GM-DC or GM-DC). CD4(+) T cell were isolated to assess lymphocyte proliferation by lymphocyte transformation test assay and the ratio of CD4(+)FOXp3(+) CD25(+) T cells were determined by fluorescene-activated cell sorter. T cell proliferation responses in GM-DC showed a significance antigen-specific proliferative response comparing with TGFbeta-GM -DC. T Cell proliferation was inhibited in co-culture system containing DC-treated TGF-beta. It can be suggested that the expsansion of T regulatory by TGF-beta-GM-DC provides a means for antigen specific control of unwanted immune reactions.

Most knowledge about dendritic cells (DCs) and regulatory T cells in humans has been gathered from circulating cells but little is known about their frequency and distribution in lymphoid organs. This report shows the frequency, phenotype and location of DCs and regulatory T cells in deceased organ donors' spleens. As determined by flow cytometry, conventional/myeloid DCs (cDCs) CD11c(high)HLA-DR(+)CD123(-/low) were 2.3 +/- 0.9% and LIN(-) HLA-DR(+)CD11c(high) 2.1 +/- 0.3% of total spleen cells. Mature CD11c(high)HLA-DR(+)CD83(+) were 1.5 +/- 0.8% and 1.0 +/- 1.6% immature CD11c(high)HLA-DR(+)CD83(-) cDC. There were 0.3 +/- 0.3% plasmacytoid DCs (pDC) CD11c(-/low)HLA-DR(+)CD123(high) and 0.3 +/- 0.1% LIN(-)HLA-DR(+)CD123(high). Cells expressing cDCs markers, BDCA-1 and BDCA-3, and pDCs markers BDCA-2 and BDCA-4 were observed in higher frequencies than DCs with other phenotypes evaluated. CD11c(+), CD123(+) and CD83(+) cells were located in subcapsular zone, T cells areas and B-cell follicles. CD4(+)CD25(high) Tregs were 0.2 +/- 0.2% and CD8(+)CD28(-) comprised 11.5 +/- 8.1% of spleen lymphocytes. FOXP3(+) cells were found in T- and B-cell areas. The improvement in cell separation, manipulation and expansion techniques, will facilitate the manipulation of donor spleen cells as a part of protocols for induction and maintenance of allograft tolerance or treatment of autoimmune diseases.

Naturally occurring CD4(+)CD25(+) T regulatory cells (nTregs) play a key role as suppressors in immune mechanisms that protect against self-destruction. The forkhead box p3 transcription factor (FOXP3) has a central role in the development of nTregs. We show here that co-culture of naïve T cells with flagellin-exposed monocyte-derived dendritic cells (MoDCs) generates CD4(+)CD25(+)FOXP3(+) T cells that transiently express FOXP3 together with CD25 but do not suppress proliferation of CD4(+)CD25(-) T cells. Moreover, purified CD4(+)CD25(+)FOXP3(+) T cells reveal a different proliferation and cytokine production profile from that of nTregs. These data indicate that in the presence of ongoing immune responses a T cell antigenic phenotype superimposable of that of nTregs does not necessarily predict suppressive function and that FOXP3 in humans is not sufficient for development and function of regulatory T cells.

Therapeutic modulation of psoriasis with targeted immunosuppressive agents defines inflammatory genes associated with disease activity and may be extrapolated to a wide range of autoimmune diseases. Cyclosporine A (CSA) is considered a "gold standard" therapy for moderate-to-severe psoriasis. We conducted a clinical trial with CSA and analyzed the treatment outcome in blood and skin of 11 responding patients. In the skin, as expected, CSA modulated genes from activated T cells and the "type 1" pathway (p40, IFN-gamma, and STAT-1-regulated genes). However, CSA also modulated genes from the newly described Th17 pathway (IL-17, IL-22, and downstream genes S100A12, DEFB-2, IL-1beta, SEPRINB3, LCN2, and CCL20). CSA also affected dendritic cells, reducing TNF and inducible NO synthase (products of inflammatory TNF- and inducible NO synthase-producing dendritic cells), CD83, and IL-23p19. We detected 220 early response genes (day 14 posttreatment) that were down-regulated by CSA. We classified >95% into proinflammatory or skin resident cells. More myeloid-derived than activated T cell genes were modulated by CSA (54 myeloid genes compared with 11 lymphocyte genes), supporting the hypothesis that myeloid derived genes contribute to pathogenic inflammation in psoriasis. In circulating mononuclear leukocytes, in stark contrast, no inflammatory gene activity was detected. Thus, we have constructed a genomic signature of successful treatment of psoriasis which may serve as a reference to guide development of other new therapies. In addition, these data also identify new gene targets for therapeutic modulation and may be applied to wide range of autoimmune diseases.

The induction of effective immune responses to tumor vaccines requires the preferential activation of effector T cells relative to regulatory or suppressive T cells. Glial tumors commonly overexpress the epidermal growth factor receptor (EGFR), which can be targeted by monoclonal antibodies. Here we show that the coating of glial tumor cells with a clinical grade anti-EGFR antibody, cetuximab, leads to enhanced tumor-specific, interferon-gamma producing CD8+ T cells by dendritic cells (DCs). The selective targeting of monoclonal antibody coated glioma cells to activating Fcgamma receptors (FcgammaRs) on DCs, which is achieved with a blocking antibody to the inhibitory form of FcgammaR, leads to the induction of antitumor immunity without the need for an exogenous maturation stimulus. Importantly, this approach reduces the concurrent induction of regulatory T cells, which can also be depleted to further enhance immunity. These data suggest that immunity to EGFR expressing tumors, including glioma, can be enhanced through the concerted function of antitumor monoclonal antibodies, activating FcgammaR, and DCs.

Foxp3+ regulatory T cells (Tregs) play a pivotal role in the maintenance of peripheral T cell tolerance and are thought to interact with dendritic cells (DC) in secondary lymphoid organs. We analyzed here the in vivo requirements for selective expansion of Ag-specific Treg vs CD4+CD25- effector T cells and engagement of Ag-specific Treg-DC interactions in secondary lymphoid organs. Using i.v. Ag delivery in the absence of inflammation, we found that CD4+CD25+Foxp3+ Tregs undergo vigorous expansion and accumulate whereas naive CD4+CD25-Foxp3- T cells undergo abortive activation. Quantifying directly the interactions between Tregs and CD11c+ DC, we found that Tregs establish cognate contacts with endogenous CD11c+ DC in spleen and lymph nodes at an early time point preceding their expansion. Importantly, we observed that as few as 10(3) Tregs selectively expanded by i.v. Ag injection are able to suppress B and T cell immune responses in mouse recipients challenged with the Ag. Our results demonstrate that Tregs are selectively mobilized by Ag recognition in the absence of inflammatory signals, and can induce thereafter potent tolerance to defined Ag targets.

Single administration of low dose cyclophosphamide (CTX) was previously reported to enhance the antitumor efficacy of immunotherapies. To investigate the possible mechanisms for this effect, we examined whether a single administration of low dose CTX could augment the immunogenicity of dendritic cell (DC) vaccines. Fifty milligrams per kilogram body weight dose of CTX was administrated intraperitoneally to mice after B16 melanoma or C26 colon carcinoma tumor models were established, DC vaccine generated from mouse bone marrow and pulsed with B16 or C26 tumor cells lysates were vaccinated 4 days later. CTX treatment potentiated the antitumor effects of the DC vaccine, and increased the proportion of IFN-gamma secreting lymphocytes in spleens. Furthermore, a significantly reduced proportion of CD4+CD25+FoxP3+ regulatory T (Treg) cells was detected by flow cytometry in spleen lymphocytes from tumor-bearing mice treated with CTX. Thus, a single administration of low dose CTX could augment antitumor immune responses of DC vaccine by reducing the proportion of CD4+CD25+FoxP3+ Treg cells in tumor-bearing mice. Our results suggested a possible mechanism of CTX-induced immunopotentiation and provided a strategy of immunotherapy combining a low dose CTX with DC vaccine.

In humans, innate immune recognition of mycobacteria, including Mycobacterium tuberculosis and bacillus Calmette-Guérin (BCG), is a feature of cells as dendritic cells (DC) and gammadelta T cells. In this study, we show that BCG infection of human monocyte-derived DC induces a rapid activation of Vgamma9Vdelta2 T cells (the major subset of gammadelta T cell pool in human peripheral blood). Indeed, in the presence of BCG-infected DC, Vgamma9Vdelta2 T cells increase both their expression of CD69 and CD25 and the production of TNF-alpha and IFN-gamma, in contrast to DC treated with Vgamma9Vdelta2 T cell-specific Ags. Without further exogenous stimuli, BCG-infected DC expand a functionally cytotoxic central memory Vgamma9Vdelta2 T cell population. This subset does not display lymph node homing receptors, but express a high amount of perforin. They are highly efficient in the killing of mycobacterial-infected primary monocytes or human monocytic THP-1 cells preserving the viability of cocultured, infected DC. This study provides further evidences about the complex relationship between important players of innate immunity and suggests an immunoregulatory role of Vgamma9Vdelta2 T cells in the control of mycobacterial infection.

Subnanomolar doses of an unaltered, naturally occurring nucleosomal histone peptide epitope, H4(71-94), when injected s.c. into lupus-prone mice, markedly prolong lifespan by generating CD4+25+ and CD8+ regulatory T cells (Treg) producing TGF-beta. The induced Treg cells suppress nuclear autoantigen-specific Th and B cells and block renal inflammation. Splenic dendritic cells (DC) captured the s.c.-injected H4(71-94) peptide rapidly and expressed a tolerogenic phenotype. The DC of the tolerized animal, especially plasmacytoid DC, produced increased amounts of TGF-beta, but diminished IL-6 on stimulation via the TLR-9 pathway by nucleosome autoantigen and other ligands; and those plasmacytoid DC blocked lupus autoimmune disease by simultaneously inducing autoantigen-specific Treg and suppressing inflammatory Th17 cells that infiltrated the kidneys of untreated lupus mice. Low-dose tolerance with H4(71-94) was effective even though the lupus immune system is spontaneously preprimed to react to the autoepitope. Thus, H4(71-94) peptide tolerance therapy that preferentially targets pathogenic autoimmune cells could spare lupus patients from chronically receiving toxic agents or global immunosuppressants and maintain remission by restoring autoantigen-specific Treg cells.

The factors that influence the functionality of human CD4(+)CD25(+) regulatory T cells are not well understood. We sought to characterize the effects of dendritic cells (DCs) on the in vitro regulatory activity of CD4(+)CD25(+) T cells obtained from peripheral blood of healthy human donors. Flow cytometry showed that a higher proportion of CD4(+)CD25(+(High)) T cells expressed surface glucocorticoid-induced tumor necrosis factor receptor family-related protein (GITR) and CTL-associated antigen 4 than CD4(+)CD25(-) or CD4(+)CD25(+(Med-low)) T cells. Intracellular Foxp3 was equivalently expressed on CD4(+)CD25(+(All)), CD4(+)CD25(+(High)), CD4(+)CD25(+(Med-low)) and CD4(+)CD25(-) T cell populations, irrespective of GITR and CTL-associated antigen 4 expression. CD4(+)CD25(+) T cells were isolated and then cultured in vitro with CD4(+)CD25(-) responder T cells and stimulated with anti-CD3 antibodies, and immature dendritic cells (iDCs), mature dendritic cells (mDCs), PBMCs or PBMCs plus anti-CD28 antibodies to provide co-stimulation. In addition, secretion of the T(h)1 cytokine IFN-gamma, IL-2 and the immunoregulatory cytokines, IL-10 and transforming growth factor (TGF)-beta, were also assessed in these cultures. We found that iDCs and mDCs were capable of reversing the suppression of proliferation mediated by CD4(+)CD25(+) regulatory T cells. However, the reversal of suppression by DCs was not dependent upon the increase of IFN-gamma and IL-2 production or inhibition of IL-10 and/or TGF-beta production. Therefore, DCs are able to reverse the suppressive effect of regulatory T cells independent of cytokine production. These results suggest for the first time that human DCs possess unique abilities which allow them to influence the functions of regulatory T cells in order to provide fine-tuning in the regulation of T cell responses.

Although standardized protocols are widely used for the generation of monocyte-derived immunostimulatory dendritic cells (DC(ims)), the inducibility of Th1 cells by DC(ims) may considerably differ. As a measure for the quality of DC(ims) generated from an individual donor at a certain time point, CD83 is used in combination with HLA-DR and CD86 to assess DC maturation. When phenotypically analyzing DC(ims), we identified a subpopulation ( approximately 60%) of CD83+, CD86+, and HLA-DR+ DC(ims) that co-expressed CD25. DC within a given DC(ims) preparation identified by lower expression of CD83 and by selective expression of CD14, however, did not co-express CD25. In order to establish CD25 as an additional maturation marker of DC(ims), we studied the DC phenotype of these cells as well as the DC-dependent T-cell proliferation and T-cell cytokine production profile after co-incubation with sorted CD25(high) and CD25(low) subpopulations of CD83+, HLA-DR+, CD86+ DC(ims). CD25(high) DC(ims) showed significant up-regulation of the DC activation molecule CD43 and induced increased levels of IL-2 secretion in allogeneic T-cells (170.7+/-86.7pg/mL) as compared to T-cells coincubated with CD25(low) DC(ims) (86.6+/-37.6pg/mL) [p=0.0224]. This was reflected by a significantly lower T-cell stimulatory capacity of CD25(low) DC(ims) (84.0% of CD25(high) DC(ims), 1:10 ratio; p=0.014) whereas the T-cell stimulatory capacity of CD25(low) DC(ims) was much higher when compared to IL-10 induced regulatory DC (55.3% of CD25(high) DC(ims); 1:10 ratio). With regard to cancer vaccination protocols, we propose to use CD25 and CD43 as additional markers for DC quality control, assessment of maturational status, and positive selection.

The B6.Sle1.Sle2.Sle3 triple congenic mouse (B6.TC) is a model of lupus coexpressing the three major NZM2410-derived susceptibility loci on a C57BL/6 background. B6.TC mice produce high titers of antinuclear nephrogenic autoantibodies and a highly penetrant glomerulonephritis. Previous studies have shown the Sle1 locus is associated with a reduced number of regulatory T cells (Treg) and that Sle3 results in intrinsic defects of myeloid cells that hyperactivate T cells. In this report, we show that B6.TC dendritic cells (DCs) accumulate in lymphoid organs and present a defective maturation process, in which bone marrow-derived, plasmacytoid, and myeloid DCs express a significantly lower level of CD80, CD86, and MHC class II. B6.TC DCs also induce a higher level of proliferation in CD4(+) T cells than B6 DCs, and B6.TC DCs block the suppressive activity of Treg. B6.TC DCs overproduce IL-6, which is necessary for the blockade of Treg activity, as shown by the effect of anti-IL-6 neutralizing Ab in the suppression assays. The overproduction of IL-6 by DCs and the blockade of Treg activity maps to Sle1, which therefore not only confers a reduced number of Treg but also blocks their ability to regulate autoreactive T cells. Taken together, these results provide a genetic and mechanistic evidence for systemic autoimmunity resulting from an impaired regulatory T cell compartment in both number and function and for Sle1-expressing DCs playing a major role in the latter defect though their production of IL-6.

Background: Previously we showed that pancreatic islets cultured for seven days in rotating bioreactors survived for >100 days in allogeneic recipients without immunosuppression. This survival coincided with almost complete elimination of "passenger" donor dendritic cells (DCs). Herein, we examined the necessity of DCs in the generation of CD4+ CD25+ T regulatory (Treg) cells., Methods: Allogeneic fresh islets or islets cultured for three days in bioreactors were transplanted to streptozotocin-induced diabetic Balb/c(stat4 -/-) as well as signal transducers and activators of transcription (Stat)4-deficient Balb/c(stat6 -/-) or Balb/c(stat4 -/-) mice. Some Balb/c recipients of fresh islet allografts were also treated with a tolerogenic protocol of anti-CD40 Ligand MR1 mAb and CTLA4Ig., Results: Islet allografts cultured for three days in bioreactors survived >100 days in all Balb/c(stat4 -/-) recipients and in 56% of Balb/c(stat6 -/-) recipients, but in none of the Balb/c recipients; the same recipients rejected fresh islet allografts. Purified T cells from long-term surviving Balb/c(stat4 -/-) recipients failed to transfer tolerance to SCID recipients of donor-type fresh islet allografts. In contrast, MR1/CTLA4Ig therapy induced tolerance to fresh islet allografts and their T cells adoptively transferred tolerance. When Balb/c or Balb/c(stat4 -/-) recipients of bioreactor-cultured islets were injected intravenously with immature syngeneic DCs, they became tolerant and developed potent alloantigen-specific CD4+ CD25+ Treg cells expressing Foxp3., Conclusion: Allogeneic islets depleted of donor DCs by culture in bioreactors have almost twofold better acceptance in Balb/c(stat4 -/-) than in Balb/c(stat6 -/-) mice, but lack Treg cells. Additional injection of host immature DCs improves tolerance in Balb/c and Balb/c(stat4 -/-) recipients by inducing potent CD4+ CD25+ Treg cells.

Dendritic cell (DC) presentation of self antigen to thymocytes is essential to the establishment of central tolerance. We show here that circulating DCs were recruited to the thymic medulla through a three-step adhesion cascade involving P-selectin, interactions of the integrin VLA-4 with its ligand VCAM-1, and pertussis toxin-sensitive chemoattractant signaling. Ovalbumin-specific OT-II thymocytes were selectively deleted after intravenous injection of antigen-loaded exogenous DCs. We documented migration of endogenous DCs to the thymus in parabiotic mice and after painting mouse skin with fluorescein isothiocyanate. Antibody to VLA-4 blocked the accumulation of peripheral tissue-derived DCs in the thymus and also inhibited the deletion of OT-II thymocytes in mice expressing membrane-bound ovalbumin in cardiac myocytes. These findings identify a migratory route by which peripheral DCs may contribute to central tolerance.

Objective: To investigate the influence of dendritic cells (DCs) on the prevalence and function of regulatory T cells (Tregs) in cytokine induced killer (CIK) cells., Methods: The blood samples of 20 patients with solid tumors were collected. The peripheral mononuclear cells (PBMCs) were isolated. CIK cells were added into the culture fluid without CD(4)(+)CD(25)(+)T cells (CIK-Treg(del) cells) and the culture fluid of regular PBMCs respectively, and the proliferation and cytotoxicity of the CIK cells were detected by BrdU method and with the cells of human lung carcinoma, breast carcinoma, colon carcinoma, and lymphoma as target cells respectively. CD(4)(+)CD(25)(+)T cells were added into another culture fluid of CIK-Treg(del) cells at the proportions of 20:1, 10:1, and 5:1 respectively, then the proliferation and cytotoxicity of the CIK cells were detected as described above. Flow cytometry was used to detect the surface markers of CIK cells. To identify the influence of DCs on the anti-tumor activity of CIK, PBMCs were isolated from the patients with solid tumor to culture the DCs and CIK cells. Dendritic cells were harvested on day 7 and co-cultured with the CIK cells (DC+CIK cells). The frequency of Tregs in CIK was determined by flow cytometry. The cytotoxicity was examined by LDH assay. The levels of TGF-beta, IL-10, IFN-gamma, IL-2, and IL-6 were analyzed by ELISA., Results: The rates of the main effector cells in CIK cells (CD(3)(+)CD(56)(+) cells) were 17% +/- 5% and 28% +/- 5% in the regular CIK cells and CIK-Treg(del) cells respectively. LDH method showed that the cytotoxicity towards tumor cells of the CIK-Treg(del) cells The rates of the main effector cells in CIK cells (CD(3)(+)CD(56)(+) cells) were 17% +/- 5% and 28% +/- 5% in the regular CIK cells and CIK-Treg(del) cells respectively. LDH method showed that the cytotoxicity towards tumor cells of the CIK-Treg(del) cells was higher than that of the regular CIK cells (P < 0.05), however, after the addition of selected cells, the cytotoxicity of the CIK-Treg(del) cells decreased. Flow cytometry showed that the proportions of CD(4)(+)CD(25)(+) Treg cells in the CIK cells and DC-CIK cells were 13% +/- 5% and 10% +/- 4% respectively (t = 3.977, P = 0.001). After the DC induction the cytotoxicity of CIK cells was significantly higher than that of the regular CIK cells. ELISA showed that after DC induction the levels of TGF-beta and IL-10 of the DC+CIK group were significantly lower than those of the regular CIK cells (t = 2.136, P = 0.046; and t = 2.965, P = 0.008), and the level of IFN-gamma was significantly higher in the DC+CIK group (t = 2.220, P = 0.039)., Conclusion: CD(4)(+)CD(25)(+) regulatory T cells inhibit the anti-tumor activity of CIK cells. The interaction between CIK cells and DCs is sufficient for the blockage of the properties of regulatory T cells. CIK cells have the desirable properties for immunotherapy approaches, especially after co-culture with DCs.