scholarly journals The surface phenotype of dendritic cells purified from mouse thymus and spleen: investigation of the CD8 expression by a subpopulation of dendritic cells.

1992 ◽  
Vol 176 (1) ◽  
pp. 47-58 ◽  
Author(s):  
D Vremec ◽  
M Zorbas ◽  
R Scollay ◽  
D J Saunders ◽  
C F Ardavin ◽  
...  
Keyword(s):  
T Cells ◽  
Dendritic Cells ◽  
T Cell ◽  
Magnetic Beads ◽  
Cell Receptor ◽  
Class Ii ◽  
Immunofluorescent Staining ◽  
Alpha Chain ◽  
Class Ii Mhc ◽  
Antigen Presenting

A new procedure for rapid isolation of dendritic cells (DC) was devised, involving collagenase digestion of tissues, dissociation of lymphoid-DC complexes, selection of light-density cells, then depletion of lymphocytes and other non-DC by treatment with a mixture of lineage-specific monoclonal antibodies (mAbs) and removal with anti-immunoglobulin-coupled magnetic beads. This enriched population (approximately 80% DC) was further purified when required by fluorescence-activated cell sorting for cells expressing high levels of class II major histocompatibility complex (MHC). The isolated DC were characterized by immunofluorescent staining using a panel of 30 mAbs. Thymic DC were surface positive for a number of markers characteristic of T cells, but they were distinct from T-lineage cells in expressing high levels of class II MHC, in lacking expression of the T cell receptor (TCR)-CD3 complex, and having TCR beta and gamma genes in germline state. Splenic DC shared many markers with thymic DC, but were negative for most T cell markers, with the exception of CD8. A substantial proportion of DC from both thymus and spleen expressed CD8 at high levels, comparable with that on T cells. This appeared to be authentic CD8, and was produced by the DC themselves, since they contained CD8 alpha mRNA. Thymic DC presented both the CD8 alpha and beta chains on the cell surface (Ly-2+3+), although the alpha chain was in excess; the splenic DC expressed only the CD8 alpha chain (Ly-2+3-). It is suggested that the expression of CD8 could endow certain antigen-presenting DC with a veto function.

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

1993 ◽  
Vol 178 (2) ◽  
pp. 633-642 ◽  
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.

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

1993 ◽  
Vol 178 (6) ◽  
pp. 1893-1901 ◽  
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.

scholarly journals Uptake of HLA Alloantigens via CD89 and CD206 Does Not Enhance Antigen Presentation by Indirect Allorecognition

2016 ◽  
Vol 2016 ◽  
pp. 1-12 ◽  
Author(s):  
Eytan Breman ◽  
Jurjen M. Ruben ◽  
Kees L. Franken ◽  
Mirjam H. M. Heemskerk ◽  
Dave L. Roelen ◽  
...  
Keyword(s):  
T Cells ◽  
Dendritic Cells ◽  
T Cell ◽  
Antigen Presentation ◽  
Antigen Presenting Cells ◽  
Mannose Receptor ◽  
Class Ii ◽  
Hla Class Ii ◽  
Indirect Allorecognition ◽  
Antigen Presenting

In organ transplantation, alloantigens are taken up by antigen presenting cells and presented via the indirect pathway to T-cells which in turn can induce allograft rejection. Monitoring of these T-cells is of major importance; however no reliable assay is available to routinely monitor indirect allorecognition. Recently we showed that HLA monomers can be successfully used to monitor indirect allorecognition. Targeting antigens to endocytic receptors on antigen presenting cells may further enhance the presentation of antigens via HLA class II and improve the efficiency of this assay. In the current study we explored targeting of HLA monomers to either CD89 expressing monocytes or mannose receptor expressing dendritic cells. Monomer-antibody complexes were generated using biotin-labeled monomers and avidin labeling of the antibodies. We demonstrate that targeting the complexes to these receptors resulted in a dose-dependent HLA class II mediated presentation to a T-cell clone. The immune-complexes were efficiently taken up and presented to T-cells. However, the level of T-cell reactivity was similar to that when only exogenous antigen was added. We conclude that HLA-A2 monomers targeted for presentation through CD89 on monocytes or mannose receptor on dendritic cells lead to proper antigen presentation but do not enhance indirect allorecognition via HLA-DR.

scholarly journals P02.02 Generating neo- and self-antigen screening libraries for class II HLA presentation

2020 ◽  
Vol 8 (Suppl 2) ◽  
pp. A20.2-A20
Author(s):  
V Pinamonti ◽  
N Felix ◽  
JM Lindner
Keyword(s):  
T Cell ◽  
T Cell Receptor ◽  
Tumor Antigens ◽  
Cell Receptor ◽  
Class Ii ◽  
Patient Specific ◽  
Spectrometric Analysis ◽  
Cross Presentation ◽  
Class Ii Mhc ◽  
Antigen Presenting

BackgroundThe identification of neo-antigens presented by tumor cells is an essential tool for cancer prevention, diagnosis, and therapy. Current approaches frequently involve mass spectrometric analysis, but these workflows do not concomitantly identify the cognate T-cell receptor. Likewise, TCR functional screens are often limited to a subset of predicted neo-epitopes.Materials and MethodsHere, we present a new method for the generation of an un-biased antigen-presenting library. Due to the genomic instability of tumors, patient-specific libraries will be cloned using random primers, ensuring the cloning of tumor-specific transcribed regions. This approach will not only address class I presentation of intracellular tumor antigens, but is also designed to simultaneously screen for cross-presentation on class II MHC complexes by professional antigen-presenting cells, an increasingly important component of anti-tumor immune responses. To guarantee presentation of genetically encoded antigens on class II MHC complexes, a signal motif for chaperone-mediated autophagy (CMA) is introduced in front of the cDNA sequence. Furthermore, antigens will be processed by the intracellular machinery, avoiding potential restrictions on spliced peptides.ConclusionsOnce established, these libraries can be exploited in high-throughput screens to functionally identify neo-antigens together with their corresponding T-cell receptor.Disclosure InformationV. Pinamonti: Other; Significant; Janssen. N. Felix: Other; Significant; Janssen. J.M. Lindner: Other; Significant; Janssen.

scholarly journals Maturational arrest from CD4+8+ to CD4+8- thymocytes in a mutant strain (LEC) of rat.

1990 ◽  
Vol 172 (6) ◽  
pp. 1615-1624 ◽  
Author(s):  
T Agui ◽  
M Oka ◽  
T Yamada ◽  
T Sakai ◽  
K Izumi ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Mutant Strain ◽  
Cell Subset ◽  
Cell Receptor ◽  
Class Ii ◽  
Class Ii Mhc ◽  
Normal Expression ◽  
Lec Rats ◽  
T Cell Maturation

A mutant strain (LEC) of rats was found to have a novel defect in T cell maturation, that is, arrest of differentiation from CD4+8+ to CD4+8- but not to CD4-8+ thymocytes. FACS analyses demonstrated a deficiency in the CD4+8- T cell subset in the thymus and a marked decrease in CD4+ T cells in peripheral lymphoid organs. Expression of the T cell receptor (TCR)/CD3 complex in CD4+8+ and CD4-8+ thymocytes of LEC rats was normal. Expression of class II major histocompatibility complex (MHC) in the thymus of LEC rats was also the same as that of normal rats. These results indicate that maturational arrest occurs only in the transition pathway from CD4+8+ to CD4+8- thymocytes, and that this mutation can not be attributed to the default of expression of either TCR/CD3, CD4, or class II MHC antigen. Consequently, dysfunction of helper T cells was observed in LEC rats, while killer T cells and B cells functioned normally. Although the complete identification of the origin of this mutation requires further studies, it is hoped that such investigations will throw light on the mechanism of positive selection.

scholarly journals Positive and Negative Selection in Transgenic Mice Expressing a T-Cell Receptor Specific for Influenza Nucleoprotein and Endogenous Superantigen

1993 ◽  
Vol 3 (3) ◽  
pp. 159-174 ◽  
Author(s):  
Clio Mamalaki ◽  
James Elliott ◽  
Trisha Norton ◽  
Nicholas Yannoutsos ◽  
Alain R. Townsend ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Transgenic Mice ◽  
T Cell Receptor ◽  
Cell Receptor ◽  
Class Ii ◽  
Cytotoxic T Cell ◽  
Dependent Manner ◽  
Class Ii Mhc ◽  
Peripheral T Cells

A transgenic mouse was generated expressing on most (>80%) of thymocytes and peripheral T cells a T-cell receptor isolated from a cytotoxic T-cell clone (F5). This clone is CD8+and recognizes αα366-374 of the nucleoprotein (NP 366-374) of influenza virus (A/NT/60/68), in the context of Class ,MHC Db(Townsend et al., 1986). The receptor utilizes the Vβ11 and Vα4 gene segments for the β chain and α chain, respectively (Palmer et al., 1989). The usage of Vβ11 makes this TcR reactive to Class II IE molecules and an endogenous ligand recently identified as a product of the endogenous mammary tumour viruses (Mtv) 8, 9, and 11 (Dyson et al., 1991). Here we report the development of F5 transgenic T cells and their function in mice of the appropriate MHC (C57BL/10 H-2b, IE-) or in mice expressing Class II MHC IE (e.g., CBA/Ca H-2kand BALB/c H-2d) and the endogenous Mtv ligands. Positive selection of CD8+T cells expressing the Vβ11 is seen in C57BL/10 transgenic mice (H-2b). Peripheral T cells from these mice are capable of killing target cells in an antigen-dependent manner after a period ofin vitroculture with IL-2. In the presence of Class II MHC IE molecules and the endogenous Mtv ligand, most of the single-positive cells carrying the transgenic T-cell receptor are absent in the thymus. Unexpectedly, CD8+peripheral T-cells in these (H-2kor H-2d) F5 mice are predominantly Vβ11 positive and also have the capacity to kill targets in an antigen-dependent manner. This is true even following backcrossing of the F5 TcR transgene to H-2dscid/scid mice, in which functional rearrangement of endogenous TcR alpha- and beta-chain genes is impaired.

scholarly journals Primary stimulation by dendritic cells induces antiviral proliferative and cytotoxic T cell responses in vitro.

1989 ◽  
Vol 169 (4) ◽  
pp. 1255-1264 ◽  
Author(s):  
S E Macatonia ◽  
P M Taylor ◽  
S C Knight ◽  
B A Askonas
Keyword(s):  
T Cells ◽  
Dendritic Cells ◽  
T Cell ◽  
T Cell Responses ◽  
Class Ii ◽  
Cytotoxic T Cell ◽  
Hanging Drop ◽  
Cell Responses ◽  
Class Ii Mhc

We used well-gassed hanging drop (20 microliters) cultures with high concentrations of purified T cells from normal BALB/c mice to examine whether dendritic cells (DC) can induce primary antiviral proliferative T cell responses and generate virus-specific CTL. We found that DC exposed to infectious influenza virus in vitro or in vivo in small numbers (0.1-1%) resulted in strong proliferation of responder T cells within 3 d, and this was strongly inhibited by antibodies to class II MHC molecules. In addition, in 5-d cultures, the influenza-treated DC generated CTL specifically able to lyse influenza-infected syngeneic target cells bearing MHC class I antigens. The most potent nucleoprotein (NP) epitope recognized by BALB/c CTL is peptide 147-158 (Arg156-) and influenza-infected DC in vitro stimulated CTL recognizing this peptide, thus mimicking the response in mice primed by intranasal influenza infection. We also induced T cell proliferation and virus-specific CTL in cultures of normal T cells by stimulating with DC pulsed with the natural NP sequence 147-158 or the potent peptide 147-158 (Arg156-). Small numbers of peritoneal exudate cells, after activation with Con A to produce class II MHC expression and after removal of DC with a specific mAb (33DI), did not lead to primary CTL generation but initiated secondary stimulation in vitro. Our results using the hanging drop culture method and DC as APC have implications for studying the T cell repertoire for viral components in humans without the necessity of previous immunization.

scholarly journals Encapsulation of Cryptococcus neoformans with Glucuronoxylomannan Inhibits the Antigen-Presenting Capacity of Monocytes

1998 ◽  
Vol 66 (2) ◽  
pp. 664-669 ◽  
Author(s):  
Cinzia Retini ◽  
Anna Vecchiarelli ◽  
Claudia Monari ◽  
Francesco Bistoni ◽  
Thomas R. Kozel
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cryptococcus Neoformans ◽  
T Cell Responses ◽  
Class Ii ◽  
Mhc Molecules ◽  
Cell Responses ◽  
Class Ii Mhc ◽  
Antigen Presenting ◽  
Cells Cultured

ABSTRACT This report examines the effect of the major capsular polysaccharide of Cryptococcus neoformans, glucuronoxylomannan (GXM), on the antigen-presenting capability of human monocytes treated with acapsular cells of C. neoformans. We found that pretreatment of acapsular cryptococci with GXM downregulates, in a dose-dependent manner, the antigen-presenting capacity of monocytes, leading to reduced proliferative T-lymphocyte responses. Similar levels of suppression occurred when monocytes were exposed to encapsulated cryptococci or acapsular cryptococci that were pretreated with GXM. The magnitude of the T-cell response correlated with the ability of monocytes to ingest the yeast. Supernatant fluids from cocultures of monocytes and T cells cultured with encapsulated cryptococci contained higher levels of interleukin-10 (IL-10) than supernatant fluids of cells with acapsular cryptococci. Addition of anti-IL-10 monoclonal antibodies to the incubation medium of monocytes and T cells cultured with encapsulated cryptococci restored proliferative T-cell responses to levels observed during culture with acapsular cryptococci. Finally, treatment of monocytes with encapsulated cryptococci or GXM-treated acapsular cryptococci suppressed expression of class II major histocompatibility complex (MHC) molecules in a manner consistent with previous reports of IL-10-mediated suppression of class II MHC molecules and suppression of proliferative T-cell responses. These results suggest a link between GXM encapsulation, increased IL-10 synthesis by monocytes, decreased expression of class II MHC molecules on monocytes, and reduced proliferative T-cell responses.

Stimulation of autologous proliferative and cytotoxic T-cell responses by “leukemic dendritic cells” derived from blast cells in acute myeloid leukemia

Blood ◽  
2001 ◽  
Vol 97 (9) ◽  
pp. 2764-2771 ◽  
Author(s):  
Beth D. Harrison ◽  
Julie A. Adams ◽  
Mark Briggs ◽  
Michelle L. Brereton ◽  
John A. Liu Yin
Keyword(s):  
T Cells ◽  
Acute Myeloid Leukemia ◽  
Dendritic Cells ◽  
T Cell ◽  
Myeloid Leukemia ◽  
T Cell Responses ◽  
Cell Responses ◽  
Antigen Presenting ◽  
Acute Myeloid ◽  
Stimulation Of

Abstract Effective presentation of tumor antigens is fundamental to strategies aimed at enrolling the immune system in eradication of residual disease after conventional treatments. Myeloid malignancies provide a unique opportunity to derive dendritic cells (DCs), functioning antigen-presenting cells, from the malignant cells themselves. These may then co-express leukemic antigens together with appropriate secondary signals and be used to generate a specific, antileukemic immune response. In this study, blasts from 40 patients with acute myeloid leukemia (AML) were cultured with combinations of granulocyte-macrophage colony-stimulating factor, interleukin 4, and tumor necrosis factor α, and development to DCs was assessed. After culture, cells from 24 samples exhibited morphological and immunophenotypic features of DCs, including expression of major histocompatibility complex class II, CD1a, CD83, and CD86, and were potent stimulators in an allogeneic mixed lymphocyte reaction (MLR). Stimulation of autologous T-cell responses was assessed by the proliferative response of autologous T cells to the leukemic DCs and by demonstration of the induction of specific, autologous, antileukemic cytotoxicity. Of 17 samples, 11 were effective stimulators in the autologous MLR, and low, but consistent, autologous, antileukemic cytotoxicity was induced in 8 of 11 cases (mean, 27%; range, 17%-37%). This study indicates that cells with enhanced antigen-presenting ability can be generated from AML blasts, that these cells can effectively prime autologous cytotoxic T cells in vitro, and that they may be used as potential vaccines in the immunotherapy of AML.

scholarly journals Response of naive antigen-specific CD4+ T cells in vitro: characteristics and antigen-presenting cell requirements.

1992 ◽  
Vol 176 (5) ◽  
pp. 1431-1437 ◽  
Author(s):  
M Croft ◽  
D D Duncan ◽  
S L Swain
Keyword(s):  
T Cells ◽  
Dendritic Cells ◽  
T Cell ◽  
Cd4 T Cells ◽  
Cd4 Cells ◽  
Peptide Antigen ◽  
Naive T Cells ◽  
Naïve T Cells ◽  
Antigen Presenting

Because of the low frequency of T cells for any particular soluble protein antigen in unprimed animals, the requirements for naive T cell responses in specific antigens have not been clearly delineated and they have been difficult to study in vitro. We have taken advantage of mice transgenic for the V beta 3/V alpha 11 T cell receptor (TCR), which can recognize a peptide of cytochrome c presented by IEk. 85-90% of CD4+ T cells in these mice express the transgenic TCR, and we show that almost all such V beta 3/V alpha 11 receptor-positive cells have a phenotype characteristic of naive T cells, including expression of high levels of CD45RB, high levels of L-selectin (Mel-14), low levels of CD44 (Pgp-1), and secretion of interleukin 2 (IL-2) as the major cytokine. Naive T cells, separated on the basis of CD45RB high expression, gave vigorous responses (proliferation and IL-2 secretion) to peptide antigen presented in vitro by a mixed antigen-presenting cell population. At least 50% of the T cell population appeared to respond, as assessed by blast transformation, entry into G1, and expression of increased levels of CD44 by 24 h. Significant contributions to the response by contaminating memory CD4+ cells were ruled out by demonstrating that the majority of the CD45RB low, L-selectin low, CD44 high cells did not express the V beta 3/V alpha 11 TCR and responded poorly to antigen. We find that proliferation and IL-2 secretion of the naive CD4 cells is minimal when resting B cells present peptide antigen, and that both splenic and bone marrow-derived macrophages are weak stimulators. Naive T cells did respond well to high numbers of activated B cells. However, dendritic cells were the most potent stimulators of proliferation and IL-2 secretion at low cell numbers, and were far superior inducers of IL-2 at higher numbers. These studies establish that naive CD4 T cells can respond vigorously to soluble antigen and indicate that maximal stimulation can be achieved by presentation of antigen on dendritic cells. This model should prove very useful in further investigations of activation requirements and functional characteristics of naive helper T cells.

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