Introduction to Treg Cells and Indicator Selection

Th1/Th2/Th17 and Treg cells are common targets for T cell analysis in flow cytometry experiments. In the previous flow cytometry guide, we discussed the introduction and indicator selection for Th1/Th2/Th17 cells. Today, our focus is on Treg cells, including the selection of detection indicators and example analyses.

1. What are Treg Cells?

Treg (Regulatory T cells) are crucial regulatory cells of the immune system. They play a vital role in maintaining self-tolerance and immune cell homeostasis. They are produced in the thymus and then migrate to the periphery, actively regulating the activation and proliferation of potentially self-reactive T cells in the normal body.

This regulation helps modulate the body's immune response. The most commonly used markers to identify Treg cells include CD4, CD25, CD127, and Foxp3. Treg cells play important roles in immune tolerance, autoimmune diseases, infectious diseases, organ transplantation, and cancer, among other conditions.

2. Classification and Markers of Treg Cells

Treg cells can be classified into two types based on their developmental origin:

① Natural Treg (nTreg) cells primarily develop in the thymus and include CD4+ Treg cells.

②Induced Treg (iTreg) cells, including various subtypes like Tr1 cells, Th3 cells, and CD8+ Treg cells, develop from initial CD4+ T cells (Th0) following antigen stimulation. Treg cells in the tumor microenvironment are mainly iTreg cells.

Figure 1: Developmental Pathway of Treg Cells

3. Selection of Detection Indicators for Treg Cells

How should you choose indicators for detecting Treg cells? Based on previous experimental experience, we have summarized the following recommendations:

① CD3 and CD4 should be highly expressed.

② Indicators in blue are important/low-expressing and require strong fluorochromes.

③ Choose either Foxp3 or CD127; Foxp3 is a nuclear factor, so membrane fixation (E-CK-A108) is required for detection, while CD127 is a cell surface marker and does not require membrane fixation.

4. Treg Cell Detection Cases

(1) Mouse Splenic Treg (3-color) Detection

Panel Design & Sample Tube Setup:

Flow Cytometry Antibody Information:

Figure 2: Mouse Splenic Treg (3-color) Detection Results

As shown in the figure above, lymphocyte populations were gated based on FSC/SSC, excluding aggregates. CD4+ T lymphocytes were then gated from CD4+ cells, and Treg cell populations were determined through CD25+Foxp3+.

Notes:

① Mouse Treg cells are identified as CD4+CD25+Foxp3+.

② CD4 gating is clear and does not require isotype controls, while CD25 and Foxp3 gating is less clear and requires isotype controls.

③ There may be fluorescence spillover, so a single-stain tube should be used for compensation adjustment.

④ Improper membrane fixation can result in high background and unclear cell populations.

(2) Human Peripheral Blood Treg (6-color) Detection

Panel Design & Sample Tube Setup:

Flow Cytometry Antibody Information:

Figure 3: Human Peripheral Blood Treg (6-color) Detection Results

As shown in the figure above, lymphocyte populations were gated based on CD45+ and SSC, excluding aggregates. CD4+ T lymphocytes were then gated from CD4+CD8- cells, and Treg cell populations were determined through CD25+CD127-/low.

Notes:

① Traditional methods for identifying human Treg cells through Foxp3 detection are relatively complex, and the membrane fixation step requires skilled operators. Currently, CD127 is commonly used for human Treg cell detection.

② Adding CD45 as an indicator helps gate lymphocyte populations. Directly gate lymphocytes based on CD45 and SSC, and analyze the proportion of CD4+CD25+CD127-/low cells. In normal human peripheral blood, Treg cells account for 3% to 10% of lymphocytes.

③ A single-stain tube should be used for compensation adjustment. 

That's all about the introduction of Treg cells and the selection of indicators. If you have any questions, please feel free to leave us a message below.