I. Introduction to Th Cells

T helper (Th) cells are functional immune cells differentiated from activated naive CD4⁺ T cells. They express CD4 molecules on their surface and require activation through T cell receptor recognition of MHC-II antigen-peptide complexes presented by antigen-presenting cells (APCs). Serving as the "command center" of the immune system, Th cells differentiate into multiple functional subsets including Th1, Th2, Th17, among others. Through secreting specific cytokines and direct cell-to-cell interactions, they assist B cells in antibody production, enhance cytotoxic T cell killing capacity, activate macrophages, and coordinate interactions among immune cells to maintain immune homeostasis. They are extensively involved in processes such as defense against pathogen infection, inflammatory responses, and immune regulation. Imbalances in these subsets may lead to immune-related diseases like allergies and autoimmune disorders.

II. Th Subsets and Their Differentiation Regulatory Pathways

Upon T cell receptor (TCR) stimulation, naive CD4⁺ T cells differentiate into diverse effector Th cell subsets under the influence of various cytokines and co-stimulatory signals.

Figure 1. Cytokine signalings regulate CD4⁺ Th cell differentiation[1]

1. Th1 Cells

• Function: Th1 cells participate in immune defense against intracellular bacteria and viruses by secreting IFN-γ.
• Key Differentiation Factors: IL-12 and IFN-γ are indispensable cytokines for Th1 differentiation.
• Core Regulatory Mechanism: TCR stimulation and IFN-γ-STAT1 signaling induce transcription factor T-bet expression, which both drives Th1 differentiation and suppresses Th2/Th17 differentiation.
• Feedback Loops T-bet promotes IFN-γ and IL-12Rβ2 expression, while IL-12 maintains T-bet expression via STAT4 signaling, forming positive feedback that enhances Th1 differentiation.

2. Th2 Cells

• Core Markers: The core markers of Th2 cells are the transcription factor GATA-3 and the cytokines IL-4, IL-5, and IL-13.
• Function: They combat helminth infections and promote tissue repair, while also being implicated in chronic inflammatory conditions such as asthma and allergies.
• Differentiation Pathway: IL-4 (exocrine or autocrine) binds to the IL-4R on CD4⁺ T cells, inducing GATA-3 expression via STAT6 phosphorylation to promote Th2-related cytokines production; transcription factors including TCF-1 (TCR activation), STAT5 (IL-2 initiation), and NFAT1 synergistically facilitate differentiation.
• Regulatory Balance Mechanism: GATA-3 suppresses Th1 development by silencing Th1-related genes, establishing a mutually counterbalance between Th1 and Th2 differentiation.

3. Th9 Cells

• Function: Th9 cells constitute a novel CD4⁺ T cell subset extensively involved in diverse pathophysiological processes including infection, allergy, cancer, and autoimmunity.
• Induction Conditions: In vitro induction requires the combined stimulation of "TCR + IL-4 + TGF-β", with core markers being IL-9 (highly expressed) and transcription factors IRF4 and PU.1.
• Regulatory Mechanism: IL-4-mediated STAT6 phosphorylation and TGF-β activate Smad (Smad2/3), PU.1, and IRF8, synergistically activating key transcription factors to form a transcription factor complex regulating IL-9 gene expression; STAT5 signaling, co-stimulatory signals, and multiple cytokines (IL-1, IL-25, IL-7, IL-21) collectively participate in differentiation regulation.

4. Th17 Cells

• Characteristics and Functions: Core hallmarks include transcription factor RORγt and cytokines IL-17A-F, IL-21, IL-10, IL-23, IL-22, etc. Primary functions involve defending against extracellular pathogens in mucosal sites while being implicated in chronic inflammation and autoimmune diseases.
• Differentiation and Stability: IL-6 + TGF-β serve as initiation signals for differentiation, driving Th17 cell formation via the STAT3-RORγt axis. Autocrine IL-21 and exogenous IL-23 maintain cellular lineage stability by continuously activating STAT3.
• Plasticity characteristics: TGF-β and IL-6 jointly induce the generation of "classical" Th17 cells, characterized by secretion of IL-17, IL-21, and IL-10; whereas IL-6, IL-1β, and IL-23 induce the generation of "pathogenic" Th17 cells, a subtype highly expressing IFN-γ, GM-CSF, and IL-22.
• Transcription Network: Centered around RORγt, multiple transcription factors including the IRF4/BATF complex, Runx1, and RORα synergistically regulate Th17-related gene expression while suppressing Foxp3.

5. Tfh cells

• Characteristics and Functions: Tfh cells are characterized by high expression of PD-1, CXCR5, ICOS, and the transcription factor Bcl-6. Their core function is to assist B cell activation, promote germinal center formation and high-affinity antibody production, thereby regulating humoral immune responses.
• Key Regulator: Bcl-6 is a key transcription factor for Tfh cell development, maintaining Tfh cell identity by suppressing lineage-specific transcription factors (T-bet, GATA-3, etc.) and Blimp-1 in other Th subsets. Multiple transcription factors including TCF-1 and BATF synergistically participate in regulation.
• Signaling and Cytokines: Among co-stimulatory signals, ICOS promotes Tfh differentiation, while CD28 inhibits Tfh differentiation by activating the ERK pathway. Regarding cytokines, IL-6 and IL-21 promote Tfh differentiation by activating STAT3 and inducing Bcl-6 expression, respectively. Conversely, the IL-2/STAT5 signaling pathway strongly suppresses Tfh development by inducing Blimp-1 expression.

6. Treg Cells

• Characteristics and Functions: Core markers include CD25, Foxp3, and inhibitory cytokines (IL-10, TGF-β, IL-35, etc.). Their primary function is to suppress abnormal immune responses and maintain immune tolerance.
• Subsets: Classified into thymically developed tTreg and peripherally induced iTreg; the latter requires antigen stimulation and co-induction by TGF-β and IL-2.
• Foxp3 Regulation: At the transcriptional level, Foxp3 expression is regulated by multiple signaling pathways. TCR/CD28 stimulation activates factors such as NF-κB, TGF-β activates Smad2/3 and the FoxO family, and IL-2 promotes Foxp3 expression via the STAT5 pathway. These pathways are also involved in epigenetic regulation.

III. Mouse Th Cell Subsets Differentiation Protocol

(I) Experimental Preparation

1. Animals:

Use 5-10 weeks-old female C57BL/6 mice (higher initial T cell proportion).

2. Instrumentation, Consumables, and Reagent Preparation

1） Core Instruments: Laminar flow hood, 37°C/5% CO₂ incubator, flow cytometer, high-speed magnetic cell sorting system, centrifuge, optical microscope.

2） Consumables: 48/24-well culture plates, 60 mm culture dishes, 15 mL centrifuge tubes, sterile dissection tools (scissors, forceps), dissecting needles, 2 cm × 2 cm nylon mesh (120 μm pore size), 3-10 mL syringes, flow cytometry-specific tubes/collection tubes.

3） Key Reagents:

• Base Solutions: Sterile PBS, PBS + 1% FBS (PBS+), Complete RPMI Medium (substitutable with IMDM or DMEM), Flow Cytometry (FACS) Buffer (PBS containing 1 mM EDTA, pH 8.0, 1% BSA, sterile-filtered).
• Antibodies and Magnetic Beads: Anti-CD3 antibody, anti-CD28 antibody, CD4 magnetic beads, intracellular cytokine detection antibodies (e.g., IFN-γ, IL-4, IL-17, Foxp3).
• Differentiation-related factors: hIL-2, rmIL-12, rmIL-4, hTGF-β, rmIL-6, rmIL-21, rmIL-23; blocking antibodies (anti-IL-4 antibody, anti-IFN-γ antibody).
• Supporting reagents: 1× ACK red blood cell lysis buffer, 70% ethanol, paraffin membrane, Golgi inhibitor, PMA, ionomycin.

3. Pre-coated culture plates

1) Dilute anti-CD3 and anti-CD28 antibodies with sterile PBS and coat 48/24-well plates:

• Th0, Th1, Th2, iTreg: Both at 1 μg/mL, 0.5-1 mL per well in 48-well plates.
• Th17: Anti-CD3 antibody 2 μg/mL + anti-CD28 antibody 1 μg/mL (higher anti-CD3 antibody concentration promotes Th17 differentiation).

2) After coating, seal to prevent evaporation contamination. Incubate overnight at 4°C or for 1 hour at 37°C. Wash once with sterile PBS before use.

(II) Isolation of Mouse Lymphoid Organs

1. Select 5-10 weeks-old female C57BL/6 mice. Spray the body surface with 70% ethanol for disinfection.

2. Restrain the mouse, make a longitudinal skin incision avoiding puncturing deep tissues, retract and secure the skin with forceps, and place harvested lymph nodes into a collection dish.

3. Incising the peritoneum to remove the spleen, which is also placed in the collection dish.

(III) Tissue Processing and Cell Suspension Preparation

1. Lymph nodes and spleen were processed separately: Place tissue in a 60 mm culture dish lined with nylon mesh. Homogenize using syringe plunger or grind with a glass slide to suspend cells.

2. Repeatedly pipette the suspension to break up clumps. Filter through the nylon mesh into a 15 mL centrifuge tube, add PBS+ to fill the tube, and centrifuge at 4°C and 475×g for 5 minutes.

3. Spleen cells: Discard the supernatant. Resuspend the pellet in 1 mL ice-cold 1× ACK lysis buffer. After lysis for 1 minute, slowly add 10 mL PBS+. Invert the centrifuge tube to mix, then centrifuge to wash. Lymph node cells should be resuspended directly in 2 mL PBS+.

4. For mixed samples, add the lymph node cell suspension to the spleen cell suspension and centrifuge again at 475×g for 5 minutes at 4°C.

(IV) CD4⁺ T Cell Enrichment

1. Label cells with CD4 magnetic beads: Add 15 μL magnetic beads + 85 μL PBS+ to each mouse tissue sample and incubate at 4°C for 15-30 minutes.

2. Wash with 10 mL PBS+, filter to remove debris, collect the pellet by centrifugation, resuspend in 100 μL PBS+, and perform positive selection using a high-speed magnetic sorting system.

3. Collect the positive fraction, wash with PBS+, and set aside. This step enhances subsequent sorting efficiency and yield.

(V) Initial CD4⁺ T Cell Sorting

1. Resuspend the cell pellet in a fluorescent antibody mixture containing CD62L, CD44, CD25, and CD4 (100 μL per mouse sample). Incubate at 4°C in the dark for 15–30 minutes.

2. Wash with PBS+, centrifuge, filter, and transfer to a dedicated flow cytometry tube. Store on ice in the dark until sorting.

3. Sort for the CD4⁺CD25⁻CD62L⁺CD44⁻ phenotype of primary CD4⁺ T cells. Pre-add 1–2 mL of complete RPMI or FBS to the bottom of the collection tube.

4. After sorting, wash cells with complete RPMI, count, and adjust concentration to 1×10⁶ cells/mL.

(VI) In Vitro Differentiation Induction

1. Cell Seeding

Aspirate the liquid from wells coated with anti-CD3 and anti-CD28. Wash each well once with 1 mL sterile PBS.

• 48-well plate: Add 0.5 mL complete RPMI medium to each well and seed 0.5 × 10⁶ primary CD4⁺ T cells.
• 24-well plate: Add 1.0 mL of complete RPMI medium to each well and seed with 1 × 10⁶ primary CD4⁺ T cells.

2. Addition of Subsets Differentiation Factors

Supplement cytokines and blocking antibodies according to the following formulations. For detection of drugs or other factors, add before/during/after differentiation based on experimental design:

Subpopulation	Core Differentiation Factor Combination
Th0	20 ng/mL hIL-2
Th1	15 ng/mL rmIL-12 + 20 ng/mL hIL-2 + 5 μg/ mL anti-mouse-IL-4
Th2	10 ng/mL rmIL-4 + 20 ng/mL hIL-2 + 2 μg /mL soluble anti-CD28 + 5 μg/mL anti-mouse-IFN-γ (Adding soluble anti-CD28 antibody to coated anti-CD28 antibody enhances Th2 cytokine production)
Th17	20 ng/mL rmIL-6 + 3 ng/mL hTGF-β + 10 ng/mL rmIL-23 + 5 μg/mL anti-mouse IFN-γ + 5 μg/mL anti-mouse IL-4
Tfh	20 ng/mL rmIL-6 + 20 ng/mL rmIL-21 + 5 μg/mL anti-mouse interferon-γ (IFN-γ) + 5 μg/mL anti-mouse interleukin-4 (IL-4) & 10 pg/mL anti-mouse transforming growth factor-β (TGF-β)
iTreg	15 ng/mL hTGF-β + 20 ng/mL hIL-2 + 5 μg/mL anti-mouse IFN-γ + 5 μg/mL anti-mouse IL-4

3. Culture Conditions

• Standard culture: Incubate at 37°C in a 5% CO₂ environment for 4–5 days, followed by gene expression and cytokine detection.
• Extended Culture: After 2 days of culture, remove TCR stimulation. Adjust the concentration to 1×10⁶ cells/mL using fresh medium containing 10 U/mL IL-2. Seed into uncoated wells to promote cell proliferation and enhance final yield.

(VII) Differentiation Effect Detection

1. Flow Cytometry Analysis of Cytokines

1) Stimulate cells for 4–6 hours with 10 ng/mL PMA + 1000 ng/mL Ionomycin or 1 μg/mL anti-CD3 antibody in the presence of a Golgi inhibitor.

2) Perform intracellular cytokine staining to detect signature factors of each subset (Th1→IFN-γ, Th2→IL-4, Th17→IL-17), while staining non-specific markers as negative controls. Th17 cultures require additional Foxp3 detection to exclude Treg reverse differentiation interference.

2. ELISA Detection of Cytokines

1） Collect culture supernatants and measure target protein levels using cytokine-specific ELISA kits.

2） If proliferation differences may affect results, cells may be washed, counted, and normalized before stimulation with 1 μg/mL anti-CD3 antibody for 24 hours, followed by supernatant collection. For Th2 IL-4 detection, cells must be washed to remove residual IL-4 from the initial culture.

3. Real-time Fluorescent Quantitative Polymerase Chain Reaction (Real-time PCR) for Gene Expression Detection

1） Collect cells, wash, and normalize. Stimulate with 1 μg/mL anti-CD3 antibody for 2–6 hours, then extract mRNA.

2） Detect expression levels of subset-specific genes (Th1→T-bet/IFNγ, Th2→GATA3/IL-4, Th17→RORγt/IL-17, iTreg→Foxp3).

IV.  Yeasen HiActi™ Cytokines

Recombinant proteins have been widely applied in core fields such as stem cell and organoid culture, recombinant protein therapeutics, CAR-T cell therapy, and antibody drugs. With the rapid development of the biopharmaceutical industry, the recombinant protein market has experienced explosive growth, and demand for high-end raw materials has risen year by year. To precisely match the continuously upgrading application needs in both research and industrial settings, and to address key pain points such as low protein activity and insufficient batch-to-batch stability, Yeasen Biotech has leveraged its years of R&D, production experience, and technological accumulation to build an innovative recombinant protein expression and purification platform. This platform focuses on providing high-activity recombinant protein products. Leveraging its proprietary expression and purification platform, Yeasen Biotech has developed a series of HiActi® cytokines—including IL-2, IL-4, and IL-12—for mouse Th subset cell culture. These products undergo rigorous quality control and cellular function validation to ensure high activity, purity, stability, and low endotoxin levels, helping you achieve optimal experimental results.

Product Data

Bioactivity of human IL-2

 Figure 2. Recombinant Human IL-2 stimulates proliferation of CTLL-2 cells. The specific activity is ≥ 1 × 107 IU/mg.

Bioactivity of Mouse IL-21

Figure 3. Measured by its ability to enhance IFN-gamma secretion in NK-92 human natural killer lymphoma cells. The EC50 for this effect is 0.2-2.3 ng/mL.

Ordering Information

Product name	Cat. No.	Specification
Recombinant Human IL-2 Protein (CHO)	90267ES	100μg/1mg
Recombinant Mouse IL-4 Protein	90144ES	5μg/50μg/100μg/500μg
Recombinant Mouse IL-6 Protein	90146ES	2μg/10μg/50μg/100μg/500μg
Recombinant Mouse IL-12 Protein	90193ES	10μg/50μg
Recombinant Mouse IL-21 Protein (HEK293)	90233ES	10μg/50μg/100μg/500μg
Recombinant Mouse IL-23 Protein	90226ES	10μg/50μg/100μg/1mg
Recombinant Mouse IFN-gamma Protein	91212ES	5μg/50μg/100μg/500μg
Recombinant Human TGF-beta 1/TGF-β1 Protein	91701ES	2μg/10μg/100μg

1. Sun L, Su Y, Jiao A, Wang X, Zhang B. T cells in health and disease. Signal Transduction and Targeted Therapy 2023; 8(1).