1 Introduction
Regulatory T cells (Treg) are a subpopulation of CD4+T cells Treg cells can be identified by a combination of different surface markers and the intracellular transcription factor FoxP3. Its main function is to suppress and terminate pro-inflammatory immune responses. They are thought to play critical roles in several human diseases and mouse models.
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CD4+ T cells (mouse) CD8+ T cells (mouse) Pan-T cells (mouse) 
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2 Treg cells of the spleen and lymph nodes
2.1 Cell subsets, frequencies and expression of markers
Treg cells account for 1-4% of all lymphocytes in secondary lymphoid organs of wild-type mice. On the other hand, in most TCR-engineered mouse strains, Treg cell frequency decreases to <1% due to defective development of Treg cells in the thymus. In contrast to human Treg cells, mouse Treg cells represent a more homogeneous population and are characterized by the expression of CD4, CD25 and the intracellular transcription factor FoxP3. In addition, Treg cells isolated from secondary lymphoid organs express the spleen/lymphatic receptors CCR7 and CD62L. Treg cell populations can also be differentiated according to their origin: natural Treg cells (nTreg cells) develop in the thymus, while induced Treg cells (iTreg cells) develop from naïve conventional T cells in the periphery. Both subgroups have similar phenotypes and comparable suppressive function. However, they differ in the epigenetic modification of the FoxP3 locus, which correlates with the stability of the Treg cell phenotype. (PMID:19109157,17298177,18493985).
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spleen (rat) lymph nodes (mouse) Related PDFs:
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2.2 Applications of Miltenyi Biotec for Treg cells of the spleen and lymph nodes
Miltenyi Biotec has developed special applications for working with and analyzing Treg cells.
- Multicolor flow cytometry analysis of regulatory T cells from mouse spleen
2.3 Preparation of spleen and lymph node specimens
The spleen and lymph nodes must be dissociated into a single cell suspension for many downstream applications, including isolation of cell subpopulations, cell culture, or flow cytometric analysis. With the the dissociation can be carried out fully automaticallyDissociador gentleMACS™and tissue-specific dissociation kits (e.g.Spleen dissociation kit, mouse). Alternatively, tissues can be dissociated using a manual method. See chapter for detailsFonts for mouse cells.
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sample preparation
2.4 Magnetic separation of Treg cells from spleen and lymph nodes
Miltenyi Biotec has developed several products for the magnetic separation of the different types and subsets of cells found in mouse spleens and lymph nodes.
For MACS cell separation technology details, please refer to the MACS manual chapterMagnetic Cell Separation.
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Magnetic Cell Separation
2.4.1 Isolation of Treg cells
Overview: Kits and reagents for separating Treg cells from spleen and lymph nodes
| starting material | isolation strategy | Comments | automation | Products |
| Isolation of Treg cells | ||||
| Single cell suspension from spleen/lymph nodes | Combination of non-target cell depletion followed by positive target cell selection | Final positive (Treg cells) and negative (T cells) fractions can be used for downstream assays/analysis | Sim* | Kit for Isolation of CD4+CD25+ Regulatory T Cells, Mouse |
| Pre-enrichment of Treg cells | ||||
| Single cell suspension from spleen/lymph nodes | Positive selection of target cells | CD25 isolation+cells | Sim* | CD25 MicroBead Kit, Maus |
| Single cell suspension from spleen/lymph nodes | Positive selection of target cells | Isolation of all CD4+cells | Sim* | CD4 (L3T4) MicroBeads, Maus |
| Single cell suspension from spleen/lymph nodes | Isolation by depletion of non-target cells | Exhaustion of all CD8+and non-T cells. | Sim* | CD4+ T cell isolation kit, mouse |
| *Automation options range from fully automated benchtop solutions such asautoMACS® Pro Separatorfor high-throughput platforms such asMultiMACS™ Cell24 Separator PlusorMultiMACS X. | ||||
Treg cells can be easily and quickly isolated from spleen and lymph node cell suspensionsKit for Isolation of Regulatory T Cells, Mouse.
see details
Isolation of mouse spleen Treg cells.CD4+CD25+Treg cells were isolated from a suspension of mouse spleen cells using CD4+CD25+Regulatory T cell isolation kit, one LD column and two MS columns, one MidiMACS™ separator and one MiniMACS separator. Cells were fluorescently stained with CD25-PE and CD4‑FITC (A) or anti-FoxP3-APC (B) and analyzed by flow cytometry with a MACSQuant® analyzer. Cell debris and dead cells were excluded from analysis based on propidium iodide scattering and fluorescence signals.
Isolation of mouse spleen Treg cells using CD4+CD25+Kit for Isolation of Regulatory T Cells, Mouse
CD4+CD25+Treg cells were isolated from a suspension of mouse spleen cells using CD4+CD25+Regulatory T cell isolation kit, one LD column and two MS columns, one MidiMACS™ separator and one MiniMACS separator. Cells were fluorescently stained with CD25-PE and CD4‑FITC (A) or anti-FoxP3-APC (B) and analyzed by flow cytometry with a MACSQuant® analyzer. Cell debris and dead cells were excluded from analysis based on propidium iodide scattering and fluorescence signals.
Treg cells from FoxP3 reporter mice, in which the FoxP3-positive cells co-express a fluorescent protein such as GFP or RFP, are typically isolated by cell sorting based on flow cytometry. However, magnetic enrichment prior to cell sorting is useful to increase target cell frequency. for this effectCD4-Microbeads, CD4+ T cell isolation kit, zCD25 MicroBead-Kitare the best choice.
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2.5 Characterization of Treg cells by flow cytometry
Mouse Treg cells can be distinguished from other T cells by CD4 and CD25 surface markers and specifically by the transcription factor FoxP3. Since there is no single marker to identify Treg cells, it is important to have a specific panel of markers to avoid interference from other cells during flow cytometry analysis.
Miltenyi Biotec offers a broad portfolio of conventional and recombinant productsAnticorpos REAfinity™and flow analysis kits for comprehensive analysis.
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2.5.1 Flow Cytometry Panels
Flow Cytometry Panels
| surface markings | intracellular markers | cytokines |
|---|---|---|
| General tags: | FoxP3 | IL-10 |
| CD45 | Helios | TGF-β |
| CD3 | Ki-67 | IL-35 |
| CD4 | ||
| CD25 | ||
| CD39 | ||
| CD73 | ||
| Fabric specific markings: | ||
| CD62L (e.g. secondary lymphoid organs) | ||
| CCR7 (e.g. secondary lymphoid organs) | ||
| α4β7 (e.g. gut) | ||
| CCR5 (e.g. inflammatory sites, tumor) | ||
| CCR6 | ||
| CCR3 | ||
| CCR9 (e.g. gut and gut) | ||
| CCR4 (z. B. Haut) | ||
| CCR8 | ||
| Activation dependent markers: | ||
| GITR | ||
| GARP | ||
| neuropilina | ||
| TIGIT | ||
| ICOS | ||
| CTLA-4 | ||
| PD-1 |
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primary antibodies
2.5.2 Analysis of cytokines and transcription factors
Abstract: Kits and reagents for analysis of cytokines and transcription factors in Treg cells by flow cytometry
| Use | Comments | Products |
| Intracellular FoxP3 staining | Buffer pool optimized for use withAnticorpos anti-FoxP3 | FoxP3 Färbepuffer-Set |
| Treg cell detection | Treg-Analysekits, Anti-Maus, REAfinity | |
| Detection of IL-10 secretion | Enable cell enumeration | IL-10 Secretion Assay in Mice - Detection Kits |
| Detection and enrichment of IL-10 secreting cells | IL-10 Secretion Assay in Mice - Cell Detection and Enrichment Kit (PE) |
Since the FoxP3 transcription factor is one of the most reliable markers for identifying Treg cells, intracellular staining is an important technique for analysis by flow cytometry. Miltenyi Biotec offers a ready-to-use productFoxP3 Färbepuffer-Setspecially designed to ensure effective staining with the various conjugated fluorochromesAnticorpos anti-FoxP3.
dedicated mouseTreg-Analysekits, Anti-Maus, REAfinity, which include fluorochrome-conjugated CD4, CD25, and anti-FoxP3 antibodies and the FoxP3 staining buffer set, enable the detection of Treg cells using surface (CD4 and CD25) and intracellular (FoxP3) markers.
Under certain conditions, Treg cells secrete anti-inflammatory cytokines such as IL-10. These IL-10 secreting Treg cells can be detected at the single cell level using theIL-10 Secretion Assay in Mice - Detection Kits (PE) or (APC).In addition, IL-10-secreting cells can also be enriched using theIL-10 Secretion Assay in Mice - Cell Detection and Enrichment (PE) Kit.
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2.6 Cell culture of Treg cells
2.6.1 Expansion of Treg cells
Summary: Kits and reagents for expanding Treg cells
| Use | Comments | Products |
| Special expansion kit | Provides optimized stimulation conditionsin-vitroExpansion of Treg cells | Treg expansion kit, mouse |
| Addition | IL-2 mouse, research grade |
Treg cells are a small population of cells. Therefore, cell expansion is required for downstream applications that require a larger number of cells. Miltenyi Biotec offers a coordinated combination ofIL-2 cytokineit is atTreg expansion kit, mouse, thereby providing optimized stimulation conditionsin-vitroExpansion of Treg cells. The expanded Treg cells show stable FoxP3 expression and retain the typical Treg cell phenotype.
see details
Analysis of the FoxP3 expression afterin-vitroExpansion of Treg cells. Treg cells were isolated with CD4+CD25+Kit for Isolation of Regulatory T Cells, Mouse. Isolated cells were analyzed for FoxP3 expression immediately after isolation (d0, left) and 7 (middle) and 10 days (right) afterwardsin-vitroExpansion with the Treg Expansion Kit, mouse.
Analysis of the FoxP3 expression afterin-vitroExpansion of Treg cells
Treg cells were isolated with CD4+CD25+Kit for Isolation of Regulatory T Cells, Mouse. Isolated cells were analyzed for FoxP3 expression immediately after isolation (d0, left) and 7 (middle) and 10 days (right) afterwardsin-vitroExpansion with the Treg Expansion Kit, mouse.
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cell culture FAQs
What are the markers for T regulatory cells? ›
Tregs are most commonly identified as CD3+CD4+CD25+FoxP3+ cells in both mice and humans. Additional cell surface markers include CD39, 5' Nucleotidase/CD73, CTLA-4, GITR, LAG-3, LRRC32, and Neuropilin-1.
Which of the following is the best marker for regulatory T cells? ›The Foxp3 transcription factor is considered the most reliable marker for Treg cells [48–50, 60].
What do regulatory T cells do quizlet? ›Regulatory T cells secrete anti-inflammatory cytokines to regulate immune function.
What do regulatory T cells do in the immune system? ›Regulatory T (Treg) cells are essential for maintaining peripheral tolerance, preventing autoimmune diseases and limiting chronic inflammatory diseases. However, they also limit beneficial responses by suppressing sterilizing immunity and limiting anti-tumour immunity.
What is the marker for T cell activation? ›Different T cell phenotypes are profiled for the expression of 3 activation markers: CD69 (early), CD25 (late), and HLA-DR (even later).
What markers do T cells express? ›T cells and their subsets can be defined by differential expression of cell surface markers including CD3, CD4, CD8 and CD25.
How do you check for Treg cells? ›Treg cells can be identified by a combination of different surface markers and the intracellular transcription factor FoxP3. Their main function is the suppression and termination of pro-inflammatory immune responses. They are considered to play a crucial role in several human diseases and mouse models.
How do you identify Tregs? ›The transcription factor FoxP3 has been shown to identify Tregs. However, the detection of FoxP3 requires cell permeabilization, thereby preventing isolation of viable Tregs. Subsequently, the extracellular marker CD127 was established for the identification of Tregs.
Are regulatory T cells immune suppression? ›Regulatory T cells (Tregs), either natural or induced, suppress a variety of physiological and pathological immune responses. One of the key issues for understanding Treg function is to determine how they suppress other lymphocytes at the molecular level in vivo and in vitro.
Where are regulatory T cells found? ›Regulatory T-cells develop primarily in the thymus (thymus-derived Tregs, tTregs), although they can also be differentiated in the periphery (peripherally-induced Tregs). The delineation of these two populations in the peripheral Treg compartment is difficult due to the lack of specific markers.
How many types of regulatory T cells are there? ›
Regulatory T cells [Tregs] are a critical subset of T cells that mediate peripheral tolerance. There are two types of Tregs: natural Tregs, which develop in the thymus, and induced Tregs, which are derived from naïve CD4+ T cells in the periphery.
Where Do regulatory T cells come from? ›Regulatory T cells are generated in the thymus (tTreg) and the periphery (pTreg). The majority of the Treg cells emerge in the thymus at the CD4 single positive stage when Foxp3 expression is induced (21).
What will happen if you lose your regulatory T cells? ›LOSS OF REGULATORY T-CELL FUNCTION IN AUTOIMMUNE DISEASE. The loss of dominant peripheral tolerance, which is normally controlled by Tregs, can lead to spontaneous autoimmune disease, immunopathology, metabolic disease, allergy, and loss of fetal–maternal tolerance during pregnancy.
Do regulatory T cells increase inflammation? ›Regulatory T (Treg) cells are a specialized lineage of suppressive CD4 T cells that act as critical negative regulators of inflammation in various biological contexts. Treg cells exposed to inflammatory conditions acquire strongly enhanced suppressive function.
How do regulatory T cells suppress inflammation? ›Tregs may suppress target cells via direct interaction of receptor–ligand pairs on Tregs and target cells; delivery of suppressive factors via gap junctions including cyclic adenosine monophosphate (cAMP); direct cytolysis; membrane-bound suppressive cytokines such as transforming growth factor-β (TGF-β); and/or ...
What are the 3 types of T cells activated by? ›T Cell Activation
There are three types of signals: TCR, BCR, and cytokine signals. If a cell receives all three signals, it will mature into an effector cell. If a cell only receives one of the signals (TCR or BCR), the cell will become useless.
There are 3 main types of T cells: cytotoxic, helper, and regulatory. Each of them has a different role in the immune response.
What are the two requirements for T cell activation? ›For T cell activation to be initiated, two signals are required: TCR recognition of MHC class II peptide and a simultaneous costimulatory signal delivered by the same APC.
What are the T cell development stage markers? ›Generation of T Cells
In the thymus, T cells develop their specific T cell markers, including TCR, CD3, CD4 or CD8, and CD2. T cells also undergo thymic education through positive and negative selection.
Regulatory T cells (Tregs) characterized by the expression of the master transcription factor forkhead box protein p3 (Foxp3) suppress anticancer immunity, thereby hindering protective immunosurveillance of tumours and hampering effective antitumour immune responses in tumour-bearing hosts, constitute a current ...
What is the surface marker of T cell? ›
Activated T cells express surface receptors, such as CD25 (the IL-2 receptor) and CD71 (the transferrin receptor), or co-stimulatory molecules (CD26, CD27, CD28, CD30, CD154 or CD40L, and CD134). Both surface marker expression and cell proliferation are predominately assessed by flow cytometry.
What are the markers for TH and TC cells? ›| Gene | Synonyms | Marker Type |
|---|---|---|
| CD3* | Pan T cells | |
| CD4 | Helper T cells (Th) | |
| CD8 | Cytotoxic T cells (Tc) | |
| CCR4 | CD194 | Tregs, Th2, Tc2 |
Abstract. Regulatory T cells, also known as Tregs, play a pivotal role in maintaining homeostasis of the immune system and self-tolerance. Tregs express CD3, CD4, CD25, and FOXP3 but lack CD127. CD4 and CD3 identify helper T lymphocytes, of which Tregs are a subset.
What is CD127 a marker for? ›CD127 is a useful marker for identifying memory and effector T cells. Its expression is down-modulated on Treg cells. The ligation of IL-7 with its receptor is important for stimulation of mature and immature T cells as well as immature B cell proliferation and development.