Expression and purification of the mGITR-Fc fusion protein and its effect on CD4+ T cells and dendritic cells in vitro

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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Contact-dependent delivery of IL-2 by dendritic cells to CD4 T cells in the contraction phase promotes their long-term survival

Protein & Cell ◽

10.1007/s13238-019-00662-0 ◽

2019 ◽

Vol 11 (2) ◽

pp. 108-123

Author(s):

Dan Tong ◽

Li Zhang ◽

Fei Ning ◽

Ying Xu ◽

Xiaoyu Hu ◽

...

Keyword(s):

T Cells ◽

Dendritic Cells ◽

T Cell ◽

T Cell Activation ◽

Cd4 T Cells ◽

Cell Activation ◽

Cd4 T Cell ◽

Immune Memory ◽

Term Survival

Abstract Common γ chain cytokines are important for immune memory formation. Among them, the role of IL-2 remains to be fully explored. It has been suggested that this cytokine is critically needed in the late phase of primary CD4 T cell activation. Lack of IL-2 at this stage sets for a diminished recall response in subsequent challenges. However, as IL-2 peak production is over at this point, the source and the exact mechanism that promotes its production remain elusive. We report here that resting, previously antigen-stimulated CD4 T cells maintain a minimalist response to dendritic cells after their peak activation in vitro. This subtle activation event may be induced by DCs without overt presence of antigen and appears to be stronger if IL-2 comes from the same dendritic cells. This encounter reactivates a miniature IL-2 production and leads a gene expression profile change in these previously activated CD4 T cells. The CD4 T cells so experienced show enhanced reactivation intensity upon secondary challenges later on. Although mostly relying on in vitro evidence, our work may implicate a subtle programing for CD4 T cell survival after primary activation in vivo.

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Modification of host dendritic cells by microchimerism-derived extracellular vesicles generates split tolerance

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1618364114 ◽

2017 ◽

Vol 114 (5) ◽

pp. 1099-1104 ◽

Cited By ~ 23

Author(s):

William Bracamonte-Baran ◽

Jonathan Florentin ◽

Ying Zhou ◽

Ewa Jankowska-Gan ◽

W. John Haynes ◽

...

Keyword(s):

T Cells ◽

Dendritic Cells ◽

Cd4 T Cells ◽

Extracellular Vesicle ◽

Membrane Microdomains ◽

Transplant Tolerance ◽

Split Tolerance ◽

Cross Dressing

Maternal microchimerism (MMc) has been associated with development of allospecific transplant tolerance, antitumor immunity, and cross-generational reproductive fitness, but its mode of action is unknown. We found in a murine model that MMc caused exposure to the noninherited maternal antigens in all offspring, but in some, MMc magnitude was enough to cause membrane alloantigen acquisition (mAAQ; “cross-dressing”) of host dendritic cells (DCs). Extracellular vesicle (EV)-enriched serum fractions from mAAQ+, but not from non-mAAQ, mice reproduced the DC cross-dressing phenomenon in vitro. In vivo, mAAQ was associated with increased expression of immune modulators PD-L1 (programmed death-ligand 1) and CD86 by myeloid DCs (mDCs) and decreased presentation of allopeptide+self-MHC complexes, along with increased PD-L1, on plasmacytoid DCs (pDCs). Remarkably, both serum EV-enriched fractions and membrane microdomains containing the acquired MHC alloantigens included CD86, but completely excluded PD-L1. In contrast, EV-enriched fractions and microdomains containing allopeptide+self-MHC did not exclude PD-L1. Adoptive transfer of allospecific transgenic CD4 T cells revealed a “split tolerance” status in mAAQ+mice: T cells recognizing intact acquired MHC alloantigens proliferated, whereas those responding to allopeptide+self-MHC did not. Using isolated pDCs and mDCs for in vitro culture with allopeptide+self-MHC–specific CD4 T cells, we could replicate their normal activation in non-mAAQ mice, and PD-L1–dependent anergy in mAAQ+hosts. We propose that EVs provide a physiologic link between microchimerism and split tolerance, with implications for tumor immunity, transplantation, autoimmunity, and reproductive success.

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IL-12hi Rapamycin-Conditioned Dendritic Cells Mediate IFN-γ–Dependent Apoptosis of Alloreactive CD4+ T Cells In Vitro and Reduce Lethal Graft-Versus-Host Disease

Biology of Blood and Marrow Transplantation ◽

10.1016/j.bbmt.2013.11.007 ◽

2014 ◽

Vol 20 (2) ◽

pp. 192-201 ◽

Cited By ~ 9

Author(s):

Elizabeth O. Stenger ◽

Brian R. Rosborough ◽

Lisa R. Mathews ◽

Huihui Ma ◽

Markus Y. Mapara ◽

...

Keyword(s):

T Cells ◽

Dendritic Cells ◽

Graft Versus Host Disease ◽

Cd4 T Cells ◽

Host Disease ◽

Graft Versus Host ◽

Ifn Γ

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The macrophage mannose receptor promotes uptake of ADAMTS13 by dendritic cells

Blood ◽

10.1182/blood-2011-09-377754 ◽

2012 ◽

Vol 119 (16) ◽

pp. 3828-3835 ◽

Cited By ~ 30

Author(s):

Nicoletta Sorvillo ◽

Wouter Pos ◽

Linda M. van den Berg ◽

Rob Fijnheer ◽

Luisa Martinez-Pomares ◽

...

Keyword(s):

T Cells ◽

Dendritic Cells ◽

Mhc Class Ii ◽

Cd4 T Cells ◽

Mannose Receptor ◽

Immune Recognition ◽

Binding Studies ◽

Macrophage Mannose Receptor ◽

Carbohydrate Recognition Domains

Abstract ADAMTS13 is a plasma metalloproteinase that regulates platelet adhesion and aggregation by cleaving ultra-large VWF multimers on the surfaces 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. The formation of autoantibodies depends on the activation of CD4+ T cells. This process requires immune recognition, endocytosis, and subsequent processing of ADAMTS13 into peptides that are presented on MHC class II molecules to CD4+ T cells by dendritic cells (DCs). In the present study, we investigated endocytosis of recombinant ADAMTS13 by immature monocyte-derived DCs using flow cytometry and confocal microscopy. After incubation of fluorescently labeled ADAMTS13 with DCs, significant uptake of ADAMTS13 was observed. Endocytosis of ADAMTS13 was completely blocked by the addition of EGTA and mannan. ADAMTS13 endocytosis was decreased in the presence of a blocking mAb directed toward the macrophage mannose receptor (MR). Furthermore, siRNA silencing of MR reduced the uptake of ADAMTS13 by DCs. In addition, in vitro binding studies confirmed the interaction of ADAMTS13 with the carbohydrate recognition domains of MR. The results of the present study indicate that sugar moieties on ADAMTS13 interact with MR, thereby promoting its endocytosis by APCs.

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Extract and fractions from birch bark affect stimulation of human dendritic cells and their activation of allogeneic CD4+ T cells in vitro

Planta Medica ◽

10.1055/s-0030-1264940 ◽

2010 ◽

Vol 76 (12) ◽

Author(s):

S Omarsdottir ◽

M Sigurpalsson ◽

A Eggertsdottir ◽

J Runarsson ◽

I Hardardottir ◽

...

Keyword(s):

T Cells ◽

Dendritic Cells ◽

Cd4 T Cells ◽

Birch Bark ◽

Human Dendritic Cells ◽

Stimulation Of

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Selective Modification of Antigen-Specific CD4+ T Cells by Retroviral-Mediated Gene Transfer and in Vitro Sensitization with Dendritic Cells

Clinical Immunology ◽

10.1006/clim.2002.5229 ◽

2002 ◽

Vol 104 (1) ◽

pp. 58-66 ◽

Cited By ~ 11

Author(s):

Chun-Ming Lin ◽

Fu-Hwei Wang

Keyword(s):

T Cells ◽

Dendritic Cells ◽

Gene Transfer ◽

Cd4 T Cells

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Productive Infection of Plasmacytoid Dendritic Cells with Human Immunodeficiency Virus Type 1 Is Triggered by CD40 Ligation

Journal of Virology ◽

10.1128/jvi.76.21.11033-11041.2002 ◽

2002 ◽

Vol 76 (21) ◽

pp. 11033-11041 ◽

Cited By ~ 90

Author(s):

Lawrence Fong ◽

Manuela Mengozzi ◽

Nancy W. Abbey ◽

Brian G. Herndier ◽

Edgar G. Engleman

Keyword(s):

Human Immunodeficiency Virus ◽

T Cells ◽

Dendritic Cells ◽

Viral Replication ◽

Cd4 T Cells ◽

Cd40 Ligand ◽

Immunodeficiency Virus ◽

Hiv 1

ABSTRACT Immature plasmacytoid dendritic cells are the principal alpha interferon-producing cells (IPC), responsible for primary antiviral immunity. IPC express surface molecules CD4, CCR5, and CXCR4, which are known coreceptors required for human immunodeficiency virus (HIV) infection. Here we show that IPC are susceptible to and replicate HIV type 1 (HIV-1). Importantly, viral replication is triggered upon activation of IPC with CD40 ligand, a signal physiologically delivered by CD4 T cells. Immunohistochemical staining of tonsil from HIV-infected individuals reveals HIV p24+ IPC, consistent with in vivo infection of these cells. IPC exposed in vitro to HIV produce alpha interferon, which partially inhibits viral replication. Nevertheless, IPC efficiently transmit HIV-1 to CD4 T-cells, and such transmission is also augmented by CD40 ligand activation. IPC produce RANTES/CCL5 and MIP-1α/CCL3 when exposed to HIV in vitro. IPC also induce naïve CD4 T cells to proliferate and would therefore preferentially infect these cells. These results indicate that IPC may play an important role in the dissemination of HIV.

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