Introduction to Th1 Th2 Th17 Cells and Selection of Indicators

Th1/Th2/Th17 and Treg cells are common subjects for T cell detection in flow cytometry experiments. Today, I will provide a brief introduction to Th1/Th2/Th17 cells and explain their detection indicators and examples of analysis.

Schematic Diagram of Th Cell Classification (Image source: "Medical Immunology")

Th cells, also known as helper T cells, primarily activate macrophages and other immune cells to engulf and eliminate antigens through different subgroups and interactions. Th cells can secrete various cytokines and participate in various immune reactions within the body. Th1/Th2/Th17 cells all belong to Th cells and express CD4+, hence they are also referred to as CD4+ T cells.

I. Introduction to Th1/Th2/Th17 Cells

· Th1 Cells:

Th1 cells mainly secrete Th1-type cytokines such as IFN-γ, TNF-α, IL-2. These cytokines promote further proliferation of Th cells, leading to cellular immune responses. Th1 cells enhance cell-mediated immune responses through the secretion of cytokines.

· Th2 Cells:

Th2 cells can secrete cytokines such as IL-4, IL-5, IL-6, IL-10, and IL-13. These cytokines promote the proliferation of Th2 cells and assist in the activation of B cells, thereby promoting B cell proliferation, differentiation, and antibody production, contributing to humoral immunity. Additionally, these cytokines can inhibit Th1 cell proliferation.

Th1/Th2 Cells and Their Secreted Cytokines (Image source: "Tuchong Creative")

· Th17 Cells:

Th17 cells participate in innate immunity and the development of certain inflammations by secreting various cytokines such as IL-17 (including IL-17A to IL-17F), IL-21, IL-22, IL-26, TNF-α. They play a significant role in immunopathological damage, especially in the occurrence and progression of autoimmune diseases.

Differentiation and Function of Th17 Cells (Image source: Wu B et al., "The TGF-beta superfamily cytokine Activin-A is induced during autoimmune neuro

II. Selection of Indicators for Th1/Th2/Th17 Cell Detection

How should indicators be selected for detecting Th1/Th2/Th17 cells? Based on previous experimental experience, I have summarized the selection criteria for you:

Th1/Th2/Th17 Cell Detection in Human Samples

1/Th2/Th17 Cell Detection in Mouse Samples

For Th1/Th2/Th17 cell detection, most literature suggests that researchers primarily choose the cytokines secreted by these cells as indicators. Some researchers may also opt to use surface markers or nuclear markers corresponding to the specific cell subgroups based on their experimental conditions.

Additionally, it's important to note that when selecting cytokines such as IFN-γ, IL-4, and IL-17A as detection indicators, stimulation blocking (E-CK-A091) and membrane permeabilization (E-CK-A109) are required. This allows the intracellular cytokines to be detected using a flow cytometer.

III. Detection Examples

Below, we'll present practical cases of detecting Th1/Th2/Th17 cells using cytokines, providing reference for everyone.

· Human Peripheral Blood Mononuclear Cell (PBMC) Th1/Th2 (4-color) Detection

Panel Setup:

Flow Cytometry Antibody Information:

Detection Results:

As shown in the image above, first, PBMC cells are gated based on FSC/SSC to exclude debris. CD3+ gating is applied to isolate the T lymphocyte population after removing cell aggregates. Th1 cell population is gated based on IFN-γ and CD4+, and Th2 cell population is gated based on IL-4 and CD4+.

· Human Peripheral Blood Mononuclear Cell (PBMC) Th17 (3-color) Detection

Panel Setup:

Flow Cytometry Antibody Information：

Detection Results:

As shown in the image above, PBMC cell population is gated based on FSC/SSC. After removing cell aggregates, CD3+ gating isolates the T lymphocyte population. Th17 cell population is determined based on CD4+ and IL-17A.

Note:

▲ After PBMC sorting, a cytokine stimulation blocking agent is required for stimulation blockade and culture (in this experiment, the reagent used is the Cytokine Activation and Protein Blockade Kit - E-CK-A091).

▲ PMA stimulation can cause partial internalization of CD4 on the surface of human T cells, so we need to use CD4 clone antibody SK3 with minimal internalization impact.

▲ IFN-γ, IL-4, and IL-17A require isotype controls since cytokine expression levels are generally not high.

▲ Membrane permeabilization agents cause significant cell damage. It is recommended to disperse the cell pellet formed after centrifugation into cell suspension before adding the membrane permeabilization agent to reduce cell damage.

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