Immunosuppressant Sinomenine Promotes Differentiation of Bone Marrow Progenitors to IL-10-Producing Ialow Regulatory Dendritic Cells Leading to the Prolongation of Allograft Survival.

Abstract Sinomenine (SN), an immunosuppressive compound derived from the Chinese medicinal plant Sinomenium acutum, effectively inhibits both proinflammatory factor production and lymphocyte proliferation. However, the effects of SN on dendritic cells (DC), the professional antigen-presenting cells, have not been elucidated. In this study, we examined the effects of SN on DC generation, maturation and function. We found that SN affected DC in a dose dependent manner; significantly inhibiting surface expression of MHC class II, CD86, and CD40, production of IL-12, TNF-a, IL-1b, and T cell-allostimulatory activity. SN targets antigen-presenting capacity of DC not only via the MHC class II pathway, but also impairs DC migration by inhibiting LPS-induced up-regulation of CCR7 and CXCR4 expression. These inhibitory effects on DC may be partially due to SN-mediated suppression of NF-kB and p38 MAPK pathways. Further study showed that SN-treated DC (DC-SN) induce donor-specific T cell hyporesponsiveness and trigger generation of IL-10-producing T regulatory-like cells in vitro. In vivo transplant studies revealed that, compared to pretreatment with immature DC, pretreatment of recipients with DC-SN could significantly prolong allograft survival, although long-term allograft acceptance was not achieved. Importantly, pretreatment of recipients with DC-SN in combination with SN administration profoundly prolonged allograft survival, resulting in long-term survival of 30% of allografts. Increased generation of CD4+CD25+ Treg cells and enhanced microchimerism in recipients brought on by combination treatment may contribute to this extension of allograft lifespan. Our results demonstrate that SN can promote bone marrow progenitors differentiation to IL-10-producing (Ialow) regulatory DC which contribute to induction of tolerance by increase generation of Treg cells.

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Small amounts of superantigen, when presented on dendritic cells, are sufficient to initiate T cell responses.

Journal of Experimental Medicine ◽

10.1084/jem.178.2.633 ◽

1993 ◽

Vol 178 (2) ◽

pp. 633-642 ◽

Cited By ~ 91

Author(s):

N Bhardwaj ◽

J W Young ◽

A J Nisanian ◽

J Baggers ◽

R M Steinman

Keyword(s):

T Cells ◽

Dendritic Cells ◽

T Cell ◽

Dendritic Cell ◽

Mhc Class Ii ◽

Cell Receptor ◽

Class Ii ◽

Cell Mediated Immunity ◽

Quiescent T Cells ◽

Antigen Presenting

Dendritic cells are potent antigen-presenting cells for several primary immune responses and therefore provide an opportunity for evaluating the amounts of cell-associated antigens that are required for inducing T cell-mediated immunity. Because dendritic cells express very high levels of major histocompatibility complex (MHC) class II products, it has been assumed that high levels of ligands bound to MHC products ("signal one") are needed to stimulate quiescent T cells. Here we describe quantitative aspects underlying the stimulation of human blood T cells by a bacterial superantigen, staphylococcal enterotoxin A (SEA). The advantages of superantigens for quantitative studies of signal one are that these ligands: (a) engage MHC class II and the T cell receptor but do not require processing; (b) are efficiently presented to large numbers of quiescent T cells; and (c) can be pulsed onto dendritic cells before their application to T cells. Thus one can relate amounts of dendritic cell-associated SEA to subsequent lymphocyte stimulation. Using radioiodinated SEA, we noted that dendritic cells can bind 30-200 times more superantigen than B cells and monocytes. Nevertheless, this high SEA binding does not underlie the strong potency of dendritic cells to present antigen to T cells. Dendritic cells can sensitize quiescent T cells, isolated using monoclonals to appropriate CD45R epitopes, after a pulse of SEA that occupies a maximum of 0.1% of surface MHC class II molecules. This corresponds to an average of 2,000 molecules per dendritic cell. At these low doses of bound SEA, monoclonal antibodies to CD3, CD4, and CD28 almost completely block T cell proliferation. In addition to suggesting new roles for MHC class II on dendritic cells, especially the capture and retention of ligands at low external concentrations, the data reveal that primary T cells can generate a response to exceptionally low levels of signal one as long as these are delivered on dendritic cells.

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Donor CD4+CD25+ T Cells Require Syngeneic MHC Class II for Initial Support of MHC-Mismatched Hematopoietic Engraftment.

Blood ◽

10.1182/blood.v104.11.251.251 ◽

2004 ◽

Vol 104 (11) ◽

pp. 251-251 ◽

Cited By ~ 1

Author(s):

Alan Hanash ◽

Robert B. Levy

Keyword(s):

Bone Marrow ◽

T Cells ◽

T Cell ◽

Mhc Class Ii ◽

Allogeneic Bone Marrow Transplantation ◽

Class Ii ◽

Allogeneic Bone ◽

Inherited Disorders ◽

Donor Chimerism

Abstract Despite the potential to cure both acquired and inherited disorders involving the hematopoietic compartment, application of allogeneic bone marrow transplantation (BMT) is limited by the frequent and severe outcome of Graft vs. Host Disease (GVHD). Unfortunately, efforts to reduce GVHD by purging the donor graft of T cells have resulted in poor engraftment and elevated disease recurrence. Alternative cell populations capable of supporting allogeneic engraftment without inducing GVHD could increase the potential for donor-recipient matching and decrease treatment associated risks. We have observed that GVHD-suppressive donor CD4+CD25+ T cells are capable of supporting allogeneic hematopoietic engraftment, as demonstrated by initial donor progenitor activity and long-term chimerism and tolerance. Using a murine MHC mismatched model transplanting 0.5–2x106 GFP+ C57BL/6 (B6) T cell-depleted bone marrow cells into 7.0 Gy sublethally irradiated BALB/c recipients, splenic CFU assessment demonstrated that co-transplantation of 1x106 B6 CD4+CD25+ T cells lead to increased donor lineage-committed GM (p<.01) and multi-potential HPP (p<.05) progenitors seven days post-BMT compared to transplantation of BM alone. Furthermore, co-transplantation of CD4+CD25+ T cells lead to lymphoid and myeloid chimerism in peripheral blood (lineage specific mean donor chimerism ± SE: B220, 67.7±15.2 vs. 0.3±0.3; CD4, 38.3±10.5 vs.0.9±0.9; CD8, 48.3±11.0 vs. 1.0±1.0; Mac-1, 58.8±16.5 vs. 0.3±0.3) and the presence of donor GM and HPP progenitors in recipient marrow two months post-BMT (mean CFU chimerism ± SE: CFU-GM, 54.5±12.8 vs. 0.0; CFU-HPP, 63.0±17.8 vs.0.0). Donor chimerism persisted six months post-BMT and was associated with tolerance to donor and host antigens by acceptance of donor and host skin grafts >50 days post-homotopic grafting. Characterization of the initial invents of engraftment support demonstrated that augmentation of donor progenitors did not require CD4+CD25+ T cell IL-10, as co-transplantation of B6-wt and B6-IL-10−/− CD4+CD25+ T cells both significantly increased total CFU-GM (mean CFU±SE: BM alone, 657.5±248.2; BM + wt, 1972±331.5; BM + IL-10−/−, 1965±401.7; both p<.05 vs. BM alone). Assessment of the antigenic requirements for activation of progenitor support demonstrated that donor CD4+CD25+ T cells did not require alloreactivity to support progenitors, as BALB/c x B6 F1 CD4+CD25+ T cells significantly increased B6 CFU-GM in BALB/c recipients (p<.001 vs. BM alone). However, B6 CD4+CD25+ T cells failed to augment C3H/HeJ CFU-GM in BALB/c recipients (p>.05 vs. BM alone), suggesting that donor CD4+CD25+ T cells might require recognition of syngeneic MHC for progenitor support. Indeed, augmentation of donor CFU-GM was abrogated when B6 CD4+CD25+ T cells were co-transplanted with B6-MHC class II−/− marrow into BALB/c recipients (p>.05 vs. BM alone). In conclusion, donor CD4+CD25+ T cells capable of promoting long-term engraftment and tolerance do not require IL-10 for support of initial donor progenitor activity, however progenitor support does require co-transplantation with syngeneic MHC class II expressing marrow. Donor CD4+CD25+ T cells may thus represent a useful alternative to unfractionated T cells for promotion of engraftment following allogeneic hematopoietic transplantation.

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C-Type Lectin Receptor Mediated Immune Recognition of ADAMTS13 Promotes HLA-DRB1*11 Dependent Presentation of CUB1-2 Derived Peptides by Dendritic Cells

Blood ◽

10.1182/blood.v118.21.196.196 ◽

2011 ◽

Vol 118 (21) ◽

pp. 196-196

Author(s):

Nicoletta Sorvillo ◽

Simon D van Haren ◽

Wouter Pos ◽

Eszter Herczenik ◽

Rob Fijnheer ◽

...

Keyword(s):

T Cells ◽

Dendritic Cells ◽

Mhc Class Ii ◽

Cd4 T Cells ◽

Antigen Presenting Cells ◽

Class Ii ◽

Immune Recognition ◽

Dependent Manner ◽

Sirna Silencing ◽

Antigen Presenting

Abstract Abstract 196 ADAMTS13 is a plasma metalloproteinase that regulates platelet adhesion and aggregation by virtue of its ability to process newly released ultra-large von Willebrand factor (VWF) multimers on the surface of endothelial cells. Autoantibodies directed against ADAMTS13 prohibit the processing of VWF multimers initiating a rare and life-threatening disorder called acquired thrombotic thrombocytopenic purpura (TTP). HLA-DRB1*11 has recently been identified as a risk factor for acquired TTP. This finding implies that formation of autoantibodies towards ADAMTS13 depends on appropriate presentation of ADAMTS13 derived peptides to CD4+ T-cells by antigen presenting cells. Here, we investigate endocytosis of recombinant ADAMTS13 by immature monocyte-derived dendritic cells (iDCs) using flow cytometry and confocal microscopy. Upon incubation of fluorescently labeled-rADAMTS13 with DCs, a time- and concentration dependent uptake of ADAMTS13 was observed. Endocytosis of ADAMTS13 was completely blocked upon addition of EGTA and mannan. We subsequently explored involvement of C-type lectins (CLRs) in the uptake of ADAMTS13 using specific blocking antibodies and siRNA silencing. We found that ADAMTS13 endocytosis was significantly decreased in cells treated with a monoclonal antibody directed towards macrophage mannose receptor (MR). Furthermore siRNA silencing of MR reduced the uptake of ADAMTS13 by dendritic cells. In vitro binding studies revealed that ADAMTS13 interacts with the carbohydrate recognition domains of MR. These data show that ADAMTS13 is internalized by iDCs in a MR-dependent manner. Antigen presenting cells continuously process endogenous and exogenous antigens into small peptides that are loaded on MHC class I or MHC class II for presentation to T lymphocytes. We have recently developed a method to analyze HLA-DR-presented peptide repertoires of dendritic cells pulsed with antigen (van Haren et al., 2011). Here, we addressed which ADAMTS13-derived peptides were presented on MHC class II alleles of a panel of both HLA-DRB1*11 positive and negative donors. Compared to previous studies with model antigens only a limited number of ADAMTS13-derived peptides were presented on MHC class II. Inspection of peptide-profiles obtained from DRB1*11 positive individuals revealed that two antigenic “core” peptides derived from the CUB1-2 domains of ADAMTS13 were presented by a DR11-positive donor. In addition to these immuno-dominant peptides several other peptides were also presented although with a markedly reduced efficiency. Our findings show that DRB1*11 expressing antigen presenting cells preferentially present antigenic “core” peptides derived from the CUB1-2 domains of ADAMTS13. We hypothesize that functional presentation of these peptides on HLA-DRB1*11 contributes to the onset of acquired TTP by stimulating low affinity self-reactive CD4+ T cells that have escaped negative selection in the thymus. Disclosures: No relevant conflicts of interest to declare.

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Immortalized dendritic cell line fully competent in antigen presentation initiates primary T cell responses in vivo.

Journal of Experimental Medicine ◽

10.1084/jem.178.6.1893 ◽

1993 ◽

Vol 178 (6) ◽

pp. 1893-1901 ◽

Cited By ~ 81

Author(s):

P Paglia ◽

G Girolomoni ◽

F Robbiati ◽

F Granucci ◽

P Ricciardi-Castagnoli

Keyword(s):

T Cells ◽

Dendritic Cells ◽

T Cell ◽

Dendritic Cell ◽

Cell Line ◽

Mhc Class Ii ◽

Class Ii ◽

Gm Csf ◽

Antigen Presenting

Dendritic cells (DC) can provide all the known costimulatory signals required for activation of unprimed T cells and are the most efficient and perhaps the critical antigen presenting cells in the induction of primary T cell-mediated immune responses. It is now shown that mouse cell lines with many of the features of DC can be generated using the MIB phi 2-N11 retroviral vector transducing a novel envAKR-mycMH2 fusion gene. The immortalized dendritic cell line (CB1) displays most of the morphologic, immunophenotypic, and functional attributes of DC, including constitutive expression of major histocompatibility complex (MHC) class II molecules, costimulatory molecules B7/BB1, heat stable antigen, intracellular adhesion molecule 1, and efficient antigen-presenting ability. Granulocyte/macrophage colony-stimulating factor (GM-CSF) proved to be effective in increasing MHC class II molecule expression and in enhancing presentation of native protein antigens. In comparison with macrophages, CB1 dendritic cells did not exhibit phagocytic and chemotactic activity in response to various stimuli and lipopolysaccharide activation was ineffective in inducing tumor necrosis factor alpha or interleukin 1 beta production. CB1 cells, pulsed with haptens in vitro and injected into naive mice were able to induce delayed-type hypersensitivity responses, further increased with pretreatment with GM-CSF, indicating that these cells may represent an immature, rather than a mature DC. The ability of CB1 to prime T cells in vivo could provide a tool to design novel immunization strategies.

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Functional comparison of spleen dendritic cells and dendritic cells cultured in vitro from bone marrow precursors

Blood ◽

10.1182/blood.v88.9.3508.bloodjournal8893508 ◽

1996 ◽

Vol 88 (9) ◽

pp. 3508-3512 ◽

Cited By ~ 71

Author(s):

K Garrigan ◽

P Moroni-Rawson ◽

C McMurray ◽

I Hermans ◽

N Abernethy ◽

...

Keyword(s):

Bone Marrow ◽

T Cells ◽

Dendritic Cells ◽

T Cell ◽

Mhc Class Ii ◽

Antigen Processing ◽

Class Ii ◽

Protein Antigen ◽

Soluble Antigen

We have compared dendritic cells (DC) isolated from mouse spleen, or generated in vitro from bone marrow (BM) precursors cultured in granulocyte macrophage-colony stimulating factor (GM-CSF) and interleukin-4 (IL-4), for the ability to process and present soluble antigen and stimulate major histocompatibility complex (MHC) Class II-restricted T cells. DC from spleen or BM cultures were equally able to stimulate the in vitro proliferation of allogeneic T cells or of antigen-specific T-cell receptor (TCR)-transgenic T cells. Both DC populations also induced comparable levels of IL-2 secretion by a T-cell hybridoma. Therefore, splenic and BM-derived DC express comparable levels of (Antigen + MHC Class II) ligands and/or costimulatory molecules and have comparable ability to stimulate T-cell responses. When presentation of a native protein antigen, rather than peptide, was evaluated, BM-derived DC were at least 50 times better than splenic DC at stimulating the proliferation of TCR-transgenic T cells. The antigen processing ability of the two populations was similar only when splenic DC were used immediately ex vivo. Therefore, unlike spleen DC, BM-derived DC maintain the capacity to process protein antigen for MHC Class II presentation during in vitro culture. Due to these characteristics, BM-derived DC may represent a useful tool in immunotherapy studies, as they combine high T-cell stimulatory properties with the capacity to process and present native antigen.

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Trypanosoma cruzi Infection Modulates In Vivo Expression of Major Histocompatibility Complex Class II Molecules on Antigen-Presenting Cells and T-Cell Stimulatory Activity of Dendritic Cells in a Strain-Dependent Manner

Infection and Immunity ◽

10.1128/iai.71.3.1194-1199.2003 ◽

2003 ◽

Vol 71 (3) ◽

pp. 1194-1199 ◽

Cited By ~ 31

Author(s):

Catalina D. Alba Soto ◽

Gerardo A. Mirkin ◽

Maria E. Solana ◽

Stella M. González Cappa

Keyword(s):

Major Histocompatibility Complex ◽

T Cell ◽

Mhc Class Ii ◽

Class Ii ◽

Peritoneal Macrophages ◽

Dependent Manner ◽

Histocompatibility Complex ◽

Antigen Presenting ◽

Strain Dependent

ABSTRACT A striking feature of Chagas' disease is the diversity of clinical presentations. Such variability may be due to the heterogeneity among Trypanosoma cruzi isolates or to the host immune response. Employing two strains which differ in their virulence, we investigated the effect of in vivo infection on professional antigen-presenting cells (APC). Acute infection with the virulent RA strain downregulated the expression of major histocompatibility complex (MHC) class II on splenic dendritic cells (DC) and inhibited its induction on peritoneal macrophages and splenic B cells. It also impaired the ability of DC to prime allogeneic T cells and to form homotypic clusters, suggesting a low maturation state of these cells. In contrast, the low-virulence K98 strain maintained the expression of MHC class II on DC or stimulated it on peritoneal macrophages and B cells and preserved DC's T-cell priming capacity and homotypic clustering. DC from RA-infected mice elicited a lower activation of T. cruzi-specific T-cell proliferation than those from K98-infected mice. APC from RA-infected mice that reached the chronic phase of infection restored MHC class II levels to those found in K98-infected mice and upregulated costimulatory molecules expression, suggesting that the immunosuppression caused by this strain is only transient. Taken together, the results indicate that in vivo infection with T. cruzi modulates APC functionality and that this is accomplished in a strain-dependent manner.

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Putative IKDCs are functionally and developmentally similar to natural killer cells, but not to dendritic cells

Journal of Experimental Medicine ◽

10.1084/jem.20071351 ◽

2007 ◽

Vol 204 (11) ◽

pp. 2579-2590 ◽

Cited By ~ 81

Author(s):

Irina Caminschi ◽

Fatma Ahmet ◽

Klaus Heger ◽

Jason Brady ◽

Stephen L. Nutt ◽

...

Keyword(s):

Dendritic Cells ◽

T Cell ◽

Nk Cells ◽

Mhc Class Ii ◽

Nk Cell ◽

Class Ii ◽

Lytic Function ◽

Plasmacytoid Dcs ◽

Antigen Presenting ◽

Developmental Features

Interferon-producing killer dendritic cells (IKDCs) have been described as possessing the lytic potential of NK cells and the antigen-presenting capacity of dendritic cells (DCs). In this study, we examine the lytic function and antigen-presenting capacity of mouse spleen IKDCs, including those found in DC preparations. IKDCs efficiently killed NK cell targets, without requiring additional activation stimuli. However, in our hands, when exposed to protein antigen or to MHC class II peptide, IKDCs induced little or no T cell proliferation relative to conventional DCs or plasmacytoid DCs, either before or after activation with CpG, or in several disease models. Certain developmental features indicated that IKDCs resembled NK cells more than DCs. IKDCs, like NK cells, did not express the transcription factor PU.1 and were absent from recombinase activating gene-2–null, common γ-chain–null (Rag2−/−Il2rg−/−) mice. When cultured with IL-15 and -18, IKDCs proliferated extensively, like NK cells. Under these conditions, a proportion of expanded IKDCs and NK cells expressed high levels of surface MHC class II. However, even such MHC class II+ IKDCs and NK cells induced poor T cell proliferative responses compared with DCs. Thus, IKDCs resemble NK cells functionally, and neither cell type could be induced to be effective antigen-presenting cells.

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CD4+CD25+Foxp3+ Regulatory T Cell Function Outside the Immune System: Differential Regulation of Hematopoietic Progenitor Cell Populations.

Blood ◽

10.1182/blood.v110.11.64.64 ◽

2007 ◽

Vol 110 (11) ◽

pp. 64-64

Author(s):

Maite Urbieta ◽

Isabel Barao ◽

Angela Panoskaltsis-Mortari ◽

Bruce R. Blazar ◽

William J. Murphy ◽

...

Keyword(s):

Bone Marrow ◽

T Cell ◽

Growth Factor ◽

Mhc Class Ii ◽

Class Ii ◽

Treg Cells ◽

Differential Regulation ◽

Innate Immune ◽

Hematopoietic Progenitor Cell ◽

Synergistic Activity

Abstract Naturally occurring CD4+CD25+Foxp3+ regulatory T (Treg) cells are responsible for a physiologically essential peripheral mechanism to maintain self tolerance. The suppressor capacity of Treg cells has been found to extend to various compartments within the host and includes the ability to modulate adaptive and innate immune responses. Interestingly, several Treg cytokines which mediate immune regulation concomitantly possess hematopoietic modulating activity. For example, transforming growth factor beta 1 (TGF-beta1) and interleukin 9 (IL-9) have been found to have opposing effects during early and late stages of myelopoiesis. We have hypothesized that the inhibition of CFU-GM observed after a co-culture period of 1–3 days with activated Tregs occurs in a contact dependent manner through a mechanism involving TGF-beta1 on the surface of the regulatory cells. Additionally, we found that Treg cells fail to inhibit CFU-GM activity from MHC class II deficient bone marrow (BM) in vitro as well as in vivo following bone marrow transplantation further supporting the notion that cognate interaction is required for this negative regulation to take place. Studies then examined Treg modulation of erythroid progenitor cells (PC). IL-9 is a T cell growth factor known to promote both growth and survival signals possessing synergistic activity together with SCF and Epo which induces enhancement of colony forming activity by erythroid PC. Anti-CD3/CD28+IL-2 activated Treg cells co-cultured with syngeneic unfractionated BM as well as lineage depleted populations resulted in a 2–3X augmentation of CFU-E vs. control cultures lacking Tegs. To examine a role for IL-9 in these observations, anti-IL-9 specific mab was added to Treg/BMC co-cultures. This antibody but not isotype control Ig abolished the enhancement of CFU-E activity. Moreover, although Tregs obtained from IL-9 deficient mice inhibited CFU-GM activity, these cells failed to enhance CFU-E. These findings indicated that IL-9 produced by Tregs is crucial for such activity and supernatants from activated Treg cultures were found to contain IL-9. Finally, Treg augmentation of CFU-E in transwell cultures using B6-wt and B6-Class II−/− lineage depleted populations showed that this enhancing activity: was not contact dependent and did not require MHC class II expression on the targeted populations. These findings reveal two distinct pathways of regulation, i.e. inhibition and enhancement as defined by cytokines, contact dependency and an MHC class II requirement. Current studies examining apoptosis and the blockade /induction of differentiation have found that Treg cells simultaneously lacking perforin and fasl are capable of mediating differential regulation indicating neither of these lytic pathways is required. In total, the present results demonstrate for the first time that Tregs can modulate responses outside the adaptive and innate immune systems.

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