Reductive Alteration of the Regulatory Function of the CD4+CD25+ T Cell Fraction in Silicosis Patients

Abstract Small numbers of human CD4+CD25+FoxP3+ Tregs can be isolated from normal peripheral blood, thus their potential clinical application is limited. In this study we tested whether Granulocyte Colony-Stimulating Factor (G-CSF)-mobilized peripheral blood stem cells (PBSC) from healthy donors can represent a useful source of CD4+CD25+ cells with regulatory activity. We utilized antibodies conjugated to microbeads (Miltenyi Biotec Inc, Auburn, CA) to immunomagnetically separate the cells on a MidiMACS device (Miltenyi) and checked the purity after isolation by flow cytometry. Starting from on average 4.0±1.8 × 108 unseparated PBSC (n=3) we positively selected 2.4±0.8 × 106 CD34+ cells (>90% purity). We then utilized the CD34− cell fraction to isolate Tregs. Due to the large content of CD4dim monocytes in the initial cell product, we initially depleted the PBSC of CD14+ cells and then utilized a two-step process that includes a CD4+ cell negative selection (using a cocktail of biotin-conjugated antibodies against CD8, CD14, CD19, CD16, CD36, CD56, CD123, TCR g/δ and Glycophorin-A) followed by a positive selection of CD25+ cells (Treg isolation kit, Miltenyi). This process allowed us to obtain 1.2±1 × 106 CD4+CD25+ cells with a purity of >70%. Intracellular expression of FoxP3 was also detected in purified CD4+CD25+ cells by flow cytometry. Primary mixed leukocyte cultures (MLC) were performed with irradiated CD34+ cells isolated from PBSC and HLA mismatched blood CD3+ responders for 6 days and T cell response was measured by a 3H-thymidine uptake assay. Tregs isolated from PBSC were added to the MLC at 1:2 Treg:responder ratio to test their regulatory function. Control experiments were performed using CD4+CD25− cells. Addition of Tregs isolated from PBSC resulted in 76±17% inhibition of anti-CD34 T cell alloreactivity (cpm: 19000±530 vs 4590±1880) (n=3), while control CD4+CD25 neg cells did not show suppressive activity. These findings show that after isolation of CD34+ cells, adequate numbers of Tregs can be obtained from the CD34− cell fraction of PBSC by using a three-step process. In addition, since Tregs isolated from PBSC suppressed in-vitro T cell alloreactivity against CD34+ cells, these findings will prompt the design of pre-clinical studies to test the combination of PBSC-derived CD34+ cells and Tregs in HLA mismatched transplantation.

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Faculty Opinions recommendation of CCR6 is expressed on an IL-10-producing, autoreactive memory T cell population with context-dependent regulatory function.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.3140956.2828054 ◽

2010 ◽

Author(s):

George Yap

Keyword(s):

T Cell ◽

Cell Population ◽

Regulatory Function ◽

Memory T Cell ◽

Context Dependent

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Indoleamine 2,3-dioxygenase specific, cytotoxic T cells as immune regulators

Blood ◽

10.1182/blood-2010-06-288498 ◽

2011 ◽

Vol 117 (7) ◽

pp. 2200-2210 ◽

Cited By ~ 69

Author(s):

Rikke Bæk Sørensen ◽

Sine Reker Hadrup ◽

Inge Marie Svane ◽

Mads Christian Hjortsø ◽

Per thor Straten ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Effector T Cells ◽

T Cell Immunity ◽

Regulatory Function ◽

Cytotoxic T Cell ◽

Interleukin 17 ◽

Patients With Cancer ◽

Indoleamine 2,3 Dioxygenase ◽

Cell Immunity

Abstract Indoleamine 2,3-dioxygenase (IDO) is an immunoregulatory enzyme that is implicated in suppressing T-cell immunity in normal and pathologic settings. Here, we describe that spontaneous cytotoxic T-cell reactivity against IDO exists not only in patients with cancer but also in healthy persons. We show that the presence of such IDO-specific CD8+ T cells boosted T-cell immunity against viral or tumor-associated antigens by eliminating IDO+ suppressive cells. This had profound effects on the balance between interleukin-17 (IL-17)–producing CD4+ T cells and regulatory T cells. Furthermore, this caused an increase in the production of the proinflammatory cytokines IL-6 and tumor necrosis factor-α while decreasing the IL-10 production. Finally, the addition of IDO-inducing agents (ie, the TLR9 ligand cytosine-phosphate-guanosine, soluble cytotoxic T lymphocyte–associated antigen 4, or interferon γ) induced IDO-specific T cells among peripheral blood mononuclear cells from patients with cancer as well as healthy donors. In the clinical setting, IDO may serve as an important and widely applicable target for immunotherapeutic strategies in which IDO plays a significant regulatory role. We describe for the first time effector T cells with a general regulatory function that may play a vital role for the mounting or maintaining of an effective adaptive immune response. We suggest terming such effector T cells “supporter T cells.”

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The Ubiquitously Expressed Csk Adaptor Protein Cbp Is Dispensable for Embryogenesis and T-Cell Development and Function

Molecular and Cellular Biology ◽

10.1128/mcb.25.23.10533-10542.2005 ◽

2005 ◽

Vol 25 (23) ◽

pp. 10533-10542 ◽

Cited By ~ 59

Author(s):

Marc-Werner Dobenecker ◽

Christian Schmedt ◽

Masato Okada ◽

Alexander Tarakhovsky

Keyword(s):

T Cell ◽

Adaptor Protein ◽

Regulatory Function ◽

Loss Of Function ◽

Thymic Development ◽

Cell Functions ◽

Carboxy Terminal ◽

And Function

ABSTRACT Regulation of Src family kinase (SFK) activity is indispensable for a functional immune system and embryogenesis. The activity of SFKs is inhibited by the presence of the carboxy-terminal Src kinase (Csk) at the cell membrane. Thus, recruitment of cytosolic Csk to the membrane-associated SFKs is crucial for its regulatory function. Previous studies utilizing in vitro and transgenic models suggested that the Csk-binding protein (Cbp), also known as phosphoprotein associated with glycosphingolipid microdomains (PAG), is the membrane adaptor for Csk. However, loss-of-function genetic evidence to support this notion was lacking. Herein, we demonstrate that the targeted disruption of the cbp gene in mice has no effect on embryogenesis, thymic development, or T-cell functions in vivo. Moreover, recruitment of Csk to the specialized membrane compartment of “lipid rafts” is not impaired by Cbp deficiency. Our results indicate that Cbp is dispensable for the recruitment of Csk to the membrane and that another Csk adaptor, yet to be discovered, compensates for the loss of Cbp.

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Endothelial stroma programs hematopoietic stem cells to differentiate into regulatory dendritic cells through IL-10

Blood ◽

10.1182/blood-2006-01-007187 ◽

2006 ◽

Vol 108 (4) ◽

pp. 1189-1197 ◽

Cited By ~ 88

Author(s):

Hua Tang ◽

Zhenhong Guo ◽

Minghui Zhang ◽

Jianli Wang ◽

Guoyou Chen ◽

...

Keyword(s):

Stem Cells ◽

Dendritic Cells ◽

T Cell ◽

Hematopoietic Stem Cells ◽

Critical Role ◽

Regulatory Function ◽

Hematopoietic Stem ◽

Regulatory Dendritic Cells

Abstract Regulatory dendritic cells (DCs) have been reported recently, but their origin is poorly understood. Our previous study demonstrated that splenic stroma can drive mature DCs to proliferate and differentiate into regulatory DCs, and their natural counterpart with similar regulatory function in normal spleens has been identified. Considering that the spleen microenvironment supports hematopoiesis and that hematopoietic stem cells (HSCs) are found in spleens of adult mice, we wondered whether splenic microenvironment could differentiate HSCs into regulatory DCs. In this report, we demonstrate that endothelial splenic stroma induce HSCs to differentiate into a distinct regulatory DC subset with high expression of CD11b but low expression of Ia. CD11bhiIalo DCs secreting high levels of TGF-β, IL-10, and NO can suppress T-cell proliferation both in vitro and in vivo. Furthermore, CD11bhiIalo DCs have the ability to potently suppress allo-DTH in vivo, indicating their preventive or therapeutic perspectives for some immunologic disorders. The inhibitory function of CD11bhiIalo DCs is mediated through NO but not through induction of regulatory T (Treg) cells or T-cell anergy. IL-10, which is secreted by endothelial splenic stroma, plays a critical role in the differentiation of the regulatory CD11bhiIalo DCs from HSCs. These results suggest that splenic microenvironment may physiologically induce regulatory DC differentiation in situ.

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The ht beta gene encodes a novel CACCC box-binding protein that regulates T-cell receptor gene expression

Molecular and Cellular Biology ◽

10.1128/mcb.13.9.5691-5701.1993 ◽

1993 ◽

Vol 13 (9) ◽

pp. 5691-5701

Author(s):

Y Wang ◽

J A Kobori ◽

L Hood

Keyword(s):

Gene Expression ◽

T Cells ◽

T Cell ◽

T Cell Receptor ◽

Binding Protein ◽

Cell Receptor ◽

Regulatory Function ◽

Human T Cell ◽

Alpha Gene ◽

Beta Gene

A gene encoding a novel CACCC box-binding protein that binds to the promoter region of the human T-cell receptor (TCR) V beta 8.1 gene and the mouse TCR alpha gene silencer has been cloned. This gene, termed ht beta, contains four zinc fingers of the class Cys2-X12-His2 that may be responsible for DNA binding and a highly negatively charged region that defines a putative transcriptional activation domain. Analysis of the expression of ht beta mRNA revealed similar expression levels and patterns in various cell lines. The bacterially expressed ht beta protein can bind to the CACCC box in both the human TCR V beta 8.1 gene promoter and the mouse TCR alpha gene silencer. The CACCC box is essential for efficient transcription of the V beta 8.1 promoter. Cotransfection with an ht beta expression plasmid and a reporter vector indicated that ht beta can activate human TCR V beta 8.1 gene transcription. ht beta also is able to counteract the silencing effect of the mouse TCR alpha gene silencer. The CACCC box has been found in almost all V beta 8.1 gene subfamily members and in both TCR alpha and beta gene enhancers in humans and mice. These results suggest that the CACCC box-binding protein may have an important regulatory function for TCR gene expression in alpha beta T cells versus gamma delta T cells.

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Loss of Diacylglycerol Kinase α Enhances Macrophage Responsiveness

Frontiers in Immunology ◽

10.3389/fimmu.2021.722469 ◽

2021 ◽

Vol 12 ◽

Author(s):

Laryssa C. Manigat ◽

Mitchell E. Granade ◽

Suchet Taori ◽

Charlotte Anne Miller ◽

Luke R. Vass ◽

...

Keyword(s):

Wound Healing ◽

T Cell ◽

Healing Process ◽

Cell Activity ◽

Regulatory Function ◽

Burned Area ◽

Wound Healing Process ◽

Wound Model ◽

Complex Wound

The diacylglycerol kinases (DGKs) are a family of enzymes responsible for the conversion of diacylglycerol (DAG) to phosphatidic acid (PA). In addition to their primary function in lipid metabolism, DGKs have recently been identified as potential therapeutic targets in multiple cancers, including glioblastoma (GBM) and melanoma. Aside from its tumorigenic properties, DGKα is also a known promoter of T-cell anergy, supporting a role as a recently-recognized T cell checkpoint. In fact, the only significant phenotype previously observed in Dgka knockout (KO) mice is the enhancement of T-cell activity. Herein we reveal a novel, macrophage-specific, immune-regulatory function of DGKα. In bone marrow-derived macrophages (BMDMs) cultured from wild-type (WT) and KO mice, we observed increased responsiveness of KO macrophages to diverse stimuli that yield different phenotypes, including LPS, IL-4, and the chemoattractant MCP-1. Knockdown (KD) of Dgka in a murine macrophage cell line resulted in similar increased responsiveness. Demonstrating in vivo relevance, we observed significantly smaller wounds in Dgka-/- mice with full-thickness cutaneous burns, a complex wound healing process in which macrophages play a key role. The burned area also demonstrated increased numbers of macrophages. In a cortical stab wound model, Dgka-/- brains show increased Iba1+ cell numbers at the needle track versus that in WT brains. Taken together, these findings identify a novel immune-regulatory checkpoint function of DGKα in macrophages with potential implications for wound healing, cancer therapy, and other settings.

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