The Purification and Functional Analysis of Human CD4<sup>+</sup>CD25<sup>high</sup> Regulatory T Cells

Baecher-Allan, Clare; Hafler, David A.

doi:10.1002/0471142735.im0704bs72

Cited by 10 publications

(9 citation statements)

References 12 publications

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“…To confirm the results of flow cytometric analysis, we separated CD25 high cells by small amounts of magnetic beads conjugated with anti-CD25 antibodies (29), obtaining a highly enriched Foxp3 ϩ population (67 to 88% Foxp3 ϩ ). These cells were incubated in medium without additional stimuli for 48 h, and IFN-␥ was measured by an immunoplex array in culture supernatants.…”

Section: Fig 3 Treg Differentiation Of Cd4 ϩ T Cells Induced By Mddc mentioning

confidence: 93%

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Modulation of the Immune and Inflammatory Responses by Plasmodium falciparum Schizont Extracts: Role of Myeloid Dendritic Cells in Effector and Regulatory Functions of CD4 ⁺ Lymphocytes

et al. 2013

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eThe optimal immune response to malaria infection comprises rapid induction of inflammatory responses promptly counteracted by regulatory mechanisms to prevent immunopathology. To evaluate the role of dendritic cells (DC) in the balance of parasite-induced inflammatory/anti-inflammatory mechanisms, we studied the activity of monocyte-derived dendritic cells (MDDC), previously exposed to soluble extracts of Plasmodium falciparum-infected red blood cells (PfSE), in the differentiation of CD4 cells isolated from donors never exposed to malaria infection. We show that MDDC exposed to PfSE are extremely efficient to induce a contemporary differentiation of TH1 effector cells and T regulatory (Treg) cells in CD4 T cells even when exposed to low concentrations of parasitic extracts. Treg cells induced by MDDC infected with PfSE (MDDC-PfSE) produce transforming growth factor beta (TGF-␤) and interleukin 10 (IL-10) and are endowed with strong suppressive properties. They also show phenotypical and functional peculiarities, such as the contemporary expression of markers of Treg and TH1 differentiation and higher sensitivity to TLR4 ligands both inducing an increasing production of suppressive cytokines. On the whole, our data indicate that MDDC exposed to PfSE orchestrate a well-balanced immune response with timely differentiation of TH1 and Treg cells in CD4 cells from nonimmune donors and suggest that, during the infection, the role of MDCC could be particularly relevant in low-parasitemia conditions.

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Section: Fig 3 Treg Differentiation Of Cd4 ϩ T Cells Induced By Mddc mentioning

confidence: 93%

“…To evaluate the suppressive activity of Treg cells induced by MDDC-PfSE, we purified CD25 high cells from CD4 ϩ cells cultured for 5 days with MDDC-PfSE, MDDC-uRBCL, or unstimulated (US) MDDC as reported above and used these cells in a functional assay as described previously (29). Briefly, CD25…”

Section: Suppressive Activity Of Treg Cells Induced By Mddc-pfsementioning

confidence: 99%

Modulation of the Immune and Inflammatory Responses by Plasmodium falciparum Schizont Extracts: Role of Myeloid Dendritic Cells in Effector and Regulatory Functions of CD4 ⁺ Lymphocytes

et al. 2013

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“…To get CD4+CD25 − T cells, CD4 line T cells were stained with anti-CD4-PerCP and anti-CD25-PE or the isotype control antibody conjugated with PE for 30 min at 4°C, CD4+CD25 − and CD4+CD25+ T cells were purified using a MoFlo high-speed cell sorter (DakoCytomation, Carpintena, CA) to a purity of >98%. In some experiments CD4+CD25 high T cells, or CD4+CD127 − CD25 high T cells were purified from PBMC or short-term CD4 lines by cell sorting using published methods (59). …”

Section: Methodsmentioning

confidence: 99%

Regulatory T Cell (Treg) Subsets Return in Patients with Refractory Lupus following Stem Cell Transplantation, and TGF-β-Producing CD8+ Treg Cells Are Associated with Immunological Remission of Lupus

Zhang

Bertucci

Ramsey‐Goldman

et al. 2009

The Journal of Immunology

154

146

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Compared with conventional drug therapy, autologous hemopoietic stem cell transplantation (HSCT) can induce very-long-term remission in refractory lupus patients. Herein, we show that in posttransplant patients, both CD4+CD25highFoxP3+ and an unusual CD8+FoxP3+ Treg subset return to levels seen in normal subjects; accompanied by almost complete inhibition of pathogenic T cell response to critical peptide autoepitopes from histones in nucleosomes, the major lupus autoantigen from apoptotic cells. In addition to a stably sustained elevation of FoxP3, posttransplant CD8 T cells also maintained markedly higher expression levels of latency-associated peptide (LAP), CD103, PD-1, PD-L1, and CTLA-4, as compared with pretransplant CD8 T cells that were identically treated by a one-time activation and rest in short-term culture. The posttransplant CD8 regulatory T cells (Treg) have autoantigen-specific and nonspecific suppressive activity, which is contact independent and predominantly TGF-β dependent. By contrast, the pretransplant CD8 T cells have helper activity, which is cell contact dependent. Although CD4+CD25high Treg cells return during clinical remission of conventional drug-treated lupus, the posttransplant patient’s CD8 Treg cells are considerably more potent, and they are absent in drug-treated patients in whom CD4 T cell autoreactivity to nucleosomal epitopes persists even during clinical remission. Therefore, unlike conventional drug therapy, hemopoietic stem cell transplantation generates a newly differentiated population of LAPhighCD103high CD8TGF-β Treg cells, which repairs the Treg deficiency in human lupus to maintain patients in true immunological remission.

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“…This type of isolation yields cells that are enriched in Treg but have low purity. We and others have used expression of CD25 to identify and isolate CD4 + CD25 high T cells which express FOXP3 and mediate suppression (Baecher-Allan and Hafler, 2006b). However, a largely arbitrary selection of high CD25 expression leads to a loss of Treg, and the number of cells obtained by this method is significantly lower than the total number of Treg present in the peripheral blood.…”

Section: Introductionmentioning

confidence: 99%

Separation of human CD4+CD39+ T cells by magnetic beads reveals two phenotypically and functionally different subsets

Schuler¹,

Harasymczuk²,

Schilling³

et al. 2011

Journal of Immunological Methods

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Objective The ectonucleotidase CD39 is an enzyme involved in adenosine production. Its surface expression on human regulatory T cells (Treg) allows for their flow-cytometry-based isolation from peripheral blood. To further develop and improve this method on a scale supporting translational studies, we introduced capture of CD39+ Treg on magnetic immunobeads. Methods Peripheral blood mononuclear cells (PBMC) obtained from healthy donors were used for negative selection of CD4+ T cells on AutoMACS using antibodies (Abs) specific for all lineage+ cells. CD4+CD39+ Treg were captured by biotin-conjugated anti-CD39 Abs and anti-biotin Ab-coated magnetic beads. Isolated CD4+CD39+ T cells were phenotyped by flow cytometry for Treg-associated markers: CD39, CD73, FOXP3, CD25, CTLA-4, CCR4, CD45RO and CD121a or for the absence of CD127 and CD49d. CFSE-based proliferation assays and ATP hydrolysis were used to measure Treg functions. Results The purity, recovery and viability of the separated CD4+CD39+ T cells were satisfactory. The isolated CD4+CD39+ T cell population consisted of FOXP3+CD25+ T cells which hydrolyzed exogenous ATP and suppressed autologous CD4+ T cell proliferation and of FOXP3negCD25neg T cells without suppressor function. The same two subsets were detectable by flow cytometry in normal PBMC, gating on CD4+CD39+, CD4+CD127neg, CD4+CD49dneg or CD4+CD25high Treg. Conclusion CD4+CD39+ Treg capture on immunobeads led to a discovery of two CD39+ subsets. Similar to CD39+ Treg in the peripheral blood, half of these cells are CD25+FOXP3+ active suppressor cells, while the other half are CD25negFOXP3neg and do not mediate suppression.

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The Purification and Functional Analysis of Human CD4⁺CD25^high Regulatory T Cells

Cited by 10 publications

References 12 publications

Modulation of the Immune and Inflammatory Responses by Plasmodium falciparum Schizont Extracts: Role of Myeloid Dendritic Cells in Effector and Regulatory Functions of CD4 ⁺ Lymphocytes

Modulation of the Immune and Inflammatory Responses by Plasmodium falciparum Schizont Extracts: Role of Myeloid Dendritic Cells in Effector and Regulatory Functions of CD4 ⁺ Lymphocytes

Regulatory T Cell (Treg) Subsets Return in Patients with Refractory Lupus following Stem Cell Transplantation, and TGF-β-Producing CD8+ Treg Cells Are Associated with Immunological Remission of Lupus

Separation of human CD4+CD39+ T cells by magnetic beads reveals two phenotypically and functionally different subsets

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The Purification and Functional Analysis of Human CD4+CD25high Regulatory T Cells

Cited by 10 publications

References 12 publications

Modulation of the Immune and Inflammatory Responses by Plasmodium falciparum Schizont Extracts: Role of Myeloid Dendritic Cells in Effector and Regulatory Functions of CD4 + Lymphocytes

Modulation of the Immune and Inflammatory Responses by Plasmodium falciparum Schizont Extracts: Role of Myeloid Dendritic Cells in Effector and Regulatory Functions of CD4 + Lymphocytes

Regulatory T Cell (Treg) Subsets Return in Patients with Refractory Lupus following Stem Cell Transplantation, and TGF-β-Producing CD8+ Treg Cells Are Associated with Immunological Remission of Lupus

Separation of human CD4+CD39+ T cells by magnetic beads reveals two phenotypically and functionally different subsets

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The Purification and Functional Analysis of Human CD4⁺CD25^high Regulatory T Cells

Modulation of the Immune and Inflammatory Responses by Plasmodium falciparum Schizont Extracts: Role of Myeloid Dendritic Cells in Effector and Regulatory Functions of CD4 ⁺ Lymphocytes

Modulation of the Immune and Inflammatory Responses by Plasmodium falciparum Schizont Extracts: Role of Myeloid Dendritic Cells in Effector and Regulatory Functions of CD4 ⁺ Lymphocytes