I. Sources and Classification of Immune Cells

1. Sources of Immune Cells

All immune cells originate from hematopoietic stem cells (HSCs) in the bone marrow. HSCs possess self-renewal capacity and multipotent differentiation potential. Through two major pathways—“myeloid differentiation” and “lymphoid differentiation”—they progressively differentiate into all types of immune cells. These cells ultimately distribute to peripheral immune organs (e.g., lymph nodes, spleen, mucosa-associated lymphoid tissue) or peripheral blood to execute immune functions.

2. Classification of Immune Cells

Immune cells form the core components of the human immune system, primarily categorized into innate immune cells and adaptive immune cells. These two cell types function complementarily: the former is responsible for rapid responses to pathogen invasion, while the latter is responsible for precise recognition and long-term memory of specific pathogens, together constituting the body's immune defense system.

Figure 1. Classification of Immune Cells

II. T Cells

1. Concept of T Cells

T lymphocytes, commonly referred to as T cells, are the core cells of the human adaptive immune system. They belong to the category of white blood cells and are named for their maturation process within the thymus (Thymus, hence the “T”). Originating from hematopoietic stem cells in the bone marrow, mature T cells reside in peripheral immune organs such as blood, lymph nodes, and the spleen. They primarily recognize pathogens (e.g., viruses, bacteria) or abnormal cells (e.g., cancer cells) through their surface “T cell antigen receptors (TCRs).”

2. Classification of T Cells

T cells are primarily classified based on function, surface markers, and activation status. Different subsets perform distinct roles in immune responses, collectively forming a precise immune defense and regulatory network. Major classifications include:

1) Classification by Core Surface Markers (CD4/CD8)

● CD8⁺ T cells: “Killer” cells that directly eliminate infected cells and cancer cells.

● CD4⁺ T cells: “Commanders” that coordinate other immune cells by secreting signaling molecules.

2) Classification by Activation State

● Naive T cells: Have not encountered specific antigens, persist long-term in peripheral immune organs, express CD45RA on their surface, and require dual signaling activation upon antigen exposure to initiate the “primary immune response” (slow onset, weak intensity).

● Effector T cells: When naive T cells are activated by antigens, they proliferate extensively and differentiate into effector T cells. These cells leave lymphoid organs and migrate to infection sites to directly eliminate pathogens.

● Memory T cells: T cells retained after the primary immune response, expressing CD45RO on their surface and capable of long-term survival. Upon re-exposure to the same antigen, they rapidly activate and proliferate into effector cells, initiating a “secondary immune response” (rapid and strong). This mechanism is central to the long-term protection provided by vaccines.

3) Classification by T Cell Receptor (TCR) Type

● αβ T cells: Comprise 90%-95% of peripheral blood T cells. Their TCR consists of α and β chains, requiring MHC molecules for antigen recognition. They form the core of adaptive immunity, responding to infections and tumors.

● γδ T cells: Only 5%-10% of peripheral blood T cells, with TCRs composed of γ and δ chains. They recognize antigens directly without MHC dependence, predominantly reside in mucosal tissues, rapidly respond to infections and tumors, and possess innate immune characteristics.

4) Classification by Immunoregulatory Function

● Helper T cells (Th): Do not directly kill cells but secrete various cytokines (e.g., IFN-γ, IL-4) to activate B cells, enhance killer cell function, and regulate cellular and humoral immunity.

● Cytotoxic T cells (CTL): Directly kill target cells by releasing perforin, granzyme, or activating the Fas/FasL pathway to eliminate virus-infected cells and cancer cells.

● Regulatory T cells (Treg): Suppress excessive immune responses by secreting IL-10, TGF-β, etc., preventing immune attacks on self-tissues and guarding against autoimmune diseases.

 Figure 2. Classification of T Cell Subpopulations [1]

3. Mechanism of T Cell

The T cell action process can be simplified into four steps: “Recognition → Activation → Execution → Memory.” The core logic involves precise target recognition followed by specialized elimination of pathogens while retaining long-term protective capabilities.

1) Antigen Recognition (Initiation)

● Antigen-presenting cells (APCs) phagocytose and process pathogens, breaking them down into antigen peptides. These peptides bind to the APC's MHC molecules, forming “peptide-MHC complexes” displayed on the cell surface.

● T cells specifically bind these complexes via their surface TCRs. Concurrently, CD4/CD8 molecules match the APC's MHC class II/I molecules, completing antigen recognition.

2) Activation and Proliferation (Preparation)

● Requires dual signaling activation: TCR binding to the complex (ensuring specificity) and B7 molecules on APCs binding to CD28 of T cells.

● Upon activation, T cells proliferate under cytokines like IL-2, differentiating into numerous effector T cells and a smaller number of memory T cells.

3) Effect Execution (Clearance)

● Cytotoxic T cells (CTL): Release perforin and granzyme, or use the Fas/FasL pathway directly kill infected/cancer cells.

● Helper T cells (Th): Secret cytokines; eg., Th1 enhances macrophage phagocytosis, Th2 promotes B cell antibody production, regulating immune responses.

● Regulatory T cells (Treg): Secret IL-10 and TGF-β to suppress excessive immune responses, preventing damage to self-tissues.

4) Memory Formation (Long-Term Protection)

● Most effector T cells undergo apoptosis after completing their task, while a minority differentiate into memory T cells. These persist long-term while retaining antigen recognition capabilities.

● Upon re-exposure to the same antigen, memory T cells rapidly differentiate into effector T cells, initiating a stronger and faster secondary immune response—the core mechanism of vaccine protection.

4. Role of T Cells in Disease

T cells play critical roles in four major disease categories: infectious diseases, autoimmune diseases, tumors, and transplant rejection. In infectious diseases, they eliminate virus-infected cells via CTLs and assist in bacterial clearance; In autoimmune diseases, dysregulation of T cell subsets triggers the immune system to attack self-tissues. In tumors, while CTLs can kill cancer cells, they are often suppressed by the tumor microenvironment. Therapies like CAR-T and TIL can modify and activate T cells for treatment. In transplant rejection, T cells attack the graft; monitoring their numbers and function enables early detection of rejection and guides adjustments to immunosuppressants, improving transplant success rates.

T cells serve as warriors against infection and sentinels against tumors, yet when uncontrolled, they become renegades attacking the self or rioters causing devastating inflammation. Modern medicine strives to “harness their strengths while mitigating their weaknesses” by modulating T cell function to treat diverse diseases. 

Figure 3. The anti- and pro-tumor immunity of γδ T cells [2]

III. Peripherally Derived T Cells

1. What Are Peripherally Derived T Cells?

Peripherally derived T cells refer to populations of mature T cells that leave the thymus and subsequently settle, proliferate, and differentiate within peripheral immune organs and tissues.

Based on surface receptors and functional characteristics, these cells can be subdivided into multiple subtypes, such as helper T cells (Th1, Th2, Th17, etc.), cytotoxic T cells (CTL), and regulatory T cells (Treg). These distinct T cell subtypes perform specialized roles and collaborate synergistically to maintain homeostasis within the body. They play pivotal roles in immune defense, immune surveillance, and immune self-regulation.

2. Cultivation of Human Peripheral-Derived T Cells

The cultivation of human peripheral-derived T cells refers to techniques that simulate the in vivo environment in vitro, enabling T cells isolated from peripheral immune tissues/body fluids to survive, proliferate, and potentially differentiate into specific functional subsets. The specific process is as follows:

Step 1: Cell Isolation and Preparation

1) Sources: Peripheral venous blood (most convenient, clinically common), umbilical cord blood, peripheral lymph nodes, or spleen tissue (requires surgical acquisition, less common).

2) Isolation of Peripheral Blood Mononuclear Cells (PBMCs): Use density gradient centrifugation (commonly Ficoll reagent) to separate red blood cells, granulocytes, etc. in whole blood from less dense mononuclear cells (including lymphocytes).

3) T cell enrichment: Employ magnetic-activated cell sorting (MACS) or flow cytometry sorting to identify T-cell surface markers (e.g., CD3), removing impurities like B cells and monocytes to obtain highly purified T cells.

Step 2: T cell activation

T cells require “dual signaling + cytokines” stimulation for activation, mimicking in vivo antigen recognition and co-stimulation processes. Common stimulation systems include:

1) First Signal (Antigen Simulation): Coat culture plates with anti-human CD3 monoclonal antibodies (e.g., OKT3) or conjugate them to magnetic beads. This mimics TCR binding to antigen peptide-MHC complexes, triggering initial T cell activation.

2) Second Signal (Co-stimulation): Add anti-human CD28 monoclonal antibody to mimic binding between B7 molecules on APCs and CD28 on T cells, ensuring full T cell activation.

3) Cytokines (Proliferation Support): Add IL-2 (core cytokine promoting T cell proliferation), IL-7/IL-15 (maintaining T cell survival and reducing apoptosis). In some scenarios, add IL-12 (inducing Th1 differentiation) or IL-4 (inducing Th2 differentiation) to regulate T cell subsets.

Step 3: In Vitro Expansion and Maintenance

1) Culture Conditions:

● Medium: RPMI 1640 or DMEM supplemented with 10% fetal bovine serum (FBS) or human serum, plus penicillin/streptomycin.

● Environment: 37°C, 5% CO₂ incubator.

2) Maintenance Culture:

● Monitor cell density and condition every 2-3 days.

● Perform a 50% medium change: aspirate half the old medium and replace with an equal volume of fresh medium and cytokines.

● Monitor cell density (maintain 1×10⁶–2×10⁶ cells/mL) to prevent nutrient depletion or apoptosis due to overcrowding.

Step 4: Detection and Analysis

After a period of culture (typically 5–14 days), cells can be analyzed:

1) Phenotypic Analysis:

● Detect cell surface markers (e.g., CD4, CD8) and intracellular cytokines via flow cytometry.

2) Functional Analysis:

● Proliferative capacity: Track proliferation using CFSE staining.

● Cytotoxicity: Assess the ability of CD8⁺ T cells to kill target cells.

● Cytokine secretion: Detect IFN-γ, IL-4, etc., via ELISPOT or flow cytometry intracellular staining.

3. Common Cytokines for Culturing Human Peripheral-Derived T Cells

In vitro culture of peripheral-derived T cells (e.g., peripheral blood T cells) relies on cytokines as core signaling molecules regulating T cell survival, activation, proliferation, differentiation, and functional maintenance. Their effects span the entire process from “resting T cell activation” to “massive expansion” and “functional phenotype maintenance.” Different cytokines precisely regulate T cell biology by specifically binding to surface receptors and activating downstream signaling pathways.

Cytokine	Core Function
IL-2	- Core proliferative factor for T cells: Activates the JAK-STAT pathway in T cells, promoting cell cycle progression. - Maintains T cell survival, inhibits activation-induced cell death (AICD). - Enhances the cytotoxic activity of CTLs and NK cells.
IL-7	- Maintains T cell homeostasis and survival: Reduces apoptosis during long-term culture. - Promotes memory T cell generation, prevents terminal exhaustion. - More favorable for naive T cell proliferation.
IL-15	- Similar proliferative effect to IL-2, but more biased towards Cytotoxic T cells (CD8⁺) and NK cells. - Induces T cell differentiation towards "Effector Memory" (TEM) phenotype, enhancing anti-tumor cytotoxicity. - Does not depend on the IL-2 receptor α chain (CD25), can activate T cells with low CD25 expression.
IL-12	- Induces T cell differentiation towards the Th1 subtype (secreting IFN-γ), enhancing cellular immune responses. - Significantly increases IFN-γ secretion by T cells and NK cells, strengthening anti-tumor/anti-viral activity. - Inhibits Th2 subtype differentiation (avoiding humoral immune bias).
IL-21	- Promotes T cell proliferation, especially enhances Follicular Helper T cell (Tfh) differentiation. - Strengthens the killing activity of CTLs and NK cells, induces expression of perforin and granzymes. - Inhibits Treg cell proliferation, reducing immune suppression.

IV. Yeasen HiActi™ Cytokines

Recombinant proteins have been extensively 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 is growing rapidly, and demand for high-end raw materials is increasing year by year. To precisely match the continuously upgrading application needs in scientific research and industrial scenarios, and to address key pain points such as low activity and insufficient batch stability, Yeasen Biotech has built an innovative recombinant protein expression and purification platform based on years of R&D, production experience, and technological accumulation, focusing 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-7, and IL-15—for human peripheral-derived T-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 4. Recombinant Human IL-2 stimulates proliferation of CTLL-2 cells. The specific activity is ≥ 1 × 10⁷ IU/mg.

Bioactivity of human IL-7

Figure 5. The ED50 as determined by a cell proliferation assay using PHA-activated human peripheral blood lymphocytes (PBL) is less than 1 ng/mL, corresponding to a specific activity of > 1 × 10⁶ IU/mg.

Bioactivity of human IL-15

Figure 6. The ED50 as determined by a cell proliferation assay using murine CTLL-2 cells is less than 0.5 ng/mL, corresponding to a specific activity of > 2.0 × 106 IU/mg.

Ordering Information

Product name	Item No.	Specification
Recombinant Human IL-2 Protein (CHO)	90267ES	100μg/1mg
Recombinant Human IL-4 Protein	90105ES	5μg/20μg/50μg/100μg/500μg
Recombinant Human IL-6 Protein	90107ES	5μg/20μg/50μg/100μg/1mg
Recombinant Human IL-7 Protein	90188ES	2μg/10μg/50μg/100μg/500μg/1mg
Recombinant Human IL-12 Protein (CHO)	90262ES	10μg/50μg/1mg
Recombinant Human IL-15 Protein	90113ES	2μg/10μg/50μg/100μg/500μg
Recombinant Human IL-21 Protein	90215ES	2μg/10μg/50μg/100μg/1mg

 

1. Maecker HT, McCoy JP, Nussenblatt R. Standardizing immunophenotyping for the Human Immunology Project. Nature Reviews Immunology 2012; 12(3):191-200.

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