CD4+ T Cell Culture and Analysis

CD4⁺ T cells play a central role in regulating adaptive immune responses against invading microbial pathogens. Naive CD4⁺ T cells are activated following recognition of an antigen-MHC complex expressed on the surface of an antigen-presenting cell (APC), coupled with co-stimulatory signals mediated by interactions between co-signaling molecules expressed on the APC and their T cell-expressed receptors.

Following activation, CD4⁺ T cells differentiate into one of several lineages of CD4⁺ T helper cell (Th) subsets depending primarily on the antigen, the strength of the TCR signal, and cytokines present in the extracellular environment. The different subsets secrete distinct combinations of cytokines, allowing the immune system to tailor its response to specific pathogens and direct the activation of other immune cell types.

This page outlines the tools we offer for studying CD4⁺ T cells, including cytokines and antibodies for CD4⁺ T cell culture, activation, and differentiation, antibodies for T helper cell characterization, and single and multianalyte immunoassays for T cell analysis.

Naïve CD4⁺ T cells are activated in a two-step process that requires:

• Recognition of an antigen/major histocompatibility complex (MHC) on an antigen-presenting cell (APC) by the T cell receptor (TCR)
• T cell co-stimulation mediated by co-stimulatory molecules on the APC interacting with specific T cell-expressed receptors

Antibodies for T Cell Activation

Anti-CD3 and anti-CD28 antibodies are frequently used in culture to mimic the two signals required for T cell activation. We offer both research use only and GMP anti-CD3 and anti-CD28 antibodies for T cell activation in vitro.

T Cell Co-Signaling

T cell activation is regulated by antigen-independent co-stimulatory and co-inhibitory signals delivered through co-signaling proteins on the APC interacting with receptors on the T cell surface. This second signal is necessary for determining the strength and duration of the T cell response including the enhancement or suppression of T cell proliferation, differentiation, and cytokine secretion. Engagement of the TCR in the absence of this second co-stimulatory signal typically results in T cell anergy or apoptosis.

As tumor cells frequently exploit inhibitory T cell co-signaling pathways to evade anti-tumor immune responses, blockade of inhibitory co-signaling pathways, as well as enhancement of stimulatory co-signaling pathways are being widely investigated as therapeutic approaches for cancer immunotherapy.

To support these studies, we offer a wide selection of blocking/neutralizing antibodies and agonist antibodies for key ligand-receptor pairs that regulate T cell activity. These antibodies are validated using the same bioassays we use to test the bioactivity of our proteins, ensuring that they block/neutralize or promote the appropriate cellular function.

Blocking/Neutralizing Antibodies for T Cell Co-Signaling Molecules

Agonist Antibodies for T Cell Co-Signaling Molecules

CTLA-4 Inhibition of B7-1/CD80-induced IL-2 Secretion and Neutralization using an Anti-Mouse CTLA-4 Antibody. IL-2 secretion induced by treating the Jurkat human acute T cell leukemia cell line with Recombinant Human B7-1/CD80 (Catalog # 10133-B1; 3 µg/mL) was inhibited by Recombinant Mouse CTLA-4 (Catalog # 434-CT) in a dose-dependent manner (orange line), as measured using the Human IL-2 Quantikine™ ELISA Kit (Catalog # D2050). Inhibition of Recombinant Human B7-1/CD80 activity by Recombinant Mouse CTLA-4 (1 µg/mL) was neutralized (blue line) by treating the cells with increasing concentrations of a Rat Anti-Mouse CTLA-4 Monoclonal Antibody (Catalog # MAB434). The ND₅₀ is typically 2.5-10 µg/mL in the presence of PHA (10 µg/mL).

Anti-Human 4-1BB Antibody Stimulates IL-2 Secretion in Human T Cells. Human T cells were treated with increasing concentrations of a Rabbit Anti-Human 4-1BB/TNFRSF9 Monoclonal Antibody (Catalog # MAB8382) in the presence of an anti-CD3 antibody, and IL-2 secretion was measured using the Human IL-2 Quantikine ELISA Kit (Catalog # D2050). The ED₅₀ for this effect is typically 1-10 µg/mL.

The different CD4⁺ T cell subsets can be identified based on their expression or lack of expression of specific cell surface receptors, transcription factors, and secreted cytokines.

Our selection of primary antibodies conjugated to one of over 30 fluorescent labels allows direct detection of cell surface and intracellular targets with the highest level of flexibility for multiplex experiments. Our antibodies are validated for specificity and reproducibility in multiple applications, including flow cytometry, immunocytochemistry/immunofluorescence (ICC/IF), immunohistochemistry (IHC), and Western blot.

When an R&D Systems antibody is not available for a specific target molecule, we recommend using an antibody from our sister brand, Novus Biologicals^™. Links provided in the tables below will take you to the Novus selection of products when appropriate.

Th1 Cells

Th1 cells are a subset of CD4⁺ T cells that is required for host defense against intracellular bacterial and viral pathogens. They differentiate from activated, naïve CD4⁺ T cells in the presence of IL-12 and IFN-γ. Th1 cells secrete high levels of the pro-inflammatory cytokines, TNF-α and IFN-γ, along with IL-2 and TNF-β, and they promote the activation and functions of M1 macrophages, natural killer cells, and CD8⁺ T cells. When not properly regulated, Th1 cells can contribute to the pathogenesis of chronic inflammatory and autoimmune diseases.

Th2 Cells

Th2 cells are a subset of CD4⁺ T cells that is required for host defense against large extracellular pathogens such as intestinal helminths and extracellular bacteria. Additionally, Th2 cells support B cell-dependent humoral immunity by secreting cytokines such as IL-4, IL-5, and IL-13, which promote B cell proliferation, differentiation of antibody-secreting plasma cells, and the production of IgE antibodies. Unregulated Th2 responses are associated with allergic inflammation and asthma.

Th9 Cells

Th9 cells are a subset of CD4⁺ T cells that is closely related to the Th2 cell subset. They primarily produce IL-9 and are involved in host defense against parasitic helminth infections. Th9 cells have been implicated in the pathogenesis of allergic asthma, inflammatory gut reactions, and various autoimmune diseases.

Th17 Cells

Th17 cells are a subset of CD4⁺ T cells that is required for host defense against specific fungal and bacterial infections. Th17 cells secrete cytokines such as IL-17A, IL-17F, IL-21, IL-22, and IL-26 (in humans) that can have both beneficial and pathogenic effects. While these cytokines stimulate the expression of secondary pro-inflammatory molecules and anti-microbial peptides, unregulated or prolonged secretion of Th17-related cytokines may contribute to chronic inflammation and tissue destruction commonly associated with autoimmune diseases.

Th22 Cells

Th22 cells are a subset of CD4⁺ T cells that is involved in maintaining the integrity of mucosal barriers. Th22 cells primarily produce IL-22, which defends against microbial pathogens and is also involved in promoting tissue repair and remodeling. High levels of IL-22 are associated with a variety of inflammatory and autoimmune diseases including psoriasis, atopic dermatitis, rheumatoid arthritis, and multiple sclerosis.

Follicular Helper T Cells

Follicular helper T (Tfh) cells are a subset of CD4⁺ T cells that is localized to the B cell follicles and germinal centers of secondary lymphoid organs, where they specialize in supporting B cell survival and differentiation. Tfh cells are required for the formation of antibody-secreting plasma cells and long-lived memory B cells, as well as affinity maturation and immunoglobulin class switching, two processes that promote the production of high-affinity antibodies against specific antigens.

Regulatory T Cells

Regulatory T cells (Tregs) are a subset of CD4⁺ T cells with immunosuppressive properties that are involved in dampening inflammation and restoring immune homeostasis. These cells produce cytokines such as IL-10, IL-35, and TGF-β, which inhibit the differentiation, proliferation, and activation of effector T cells.

Detection of Regulatory T Cells Using Multicolor Flow Cytometry. Human regulatory T cells were expanded from peripheral blood mononuclear cells (PBMCs) using a Mouse Anti-Human CD3 Monoclonal Antibody (Catalog # MAB100; 10 µg/mL) and a Goat Anti-Human CD28 Antigen Affinity-purified Polyclonal Antibody (Catalog # AF-342-PB; 5 µg/mL), plus Recombinant Human IL-2 (Catalog # 202-IL; 20 ng/mL) and Recombinant Human TGF-β (Catalog # 7754-BH; 10 ng/mL) for 2 days. The cells were stained with antibodies in the Regulatory T Cell (Treg) Flow Cytometry Panel (Catalog # FMC-P-004), which includes an Alexa Fluor^® 405-conjugated Mouse Anti-Human CD3 Monoclonal Antibody, a PerCP-conjugated Mouse Anti-Human CD4 Monoclonal Antibody, a PE-conjugated Mouse Anti-Human CD25/IL-2 R Monoclonal Antibody, an Alexa Fluor^® 700-conjugated Mouse Anti-Human CD127/IL-7 R Monoclonal Antibody, a FITC-conjugated Mouse Anti-Human GITR/TNFRSF18 Monoclonal Antibody, and an Alexa Fluor^® 647-conjugated Mouse Anti-Human FoxP3 Monoclonal Antibody. 

ELISpot Kits are highly sensitive, microplate-based assays for the detection of cytokine-secreting cells. Complete ELISpot Kits are ready-to-run and require no assay development or refinement. As an alternative to our complete kits, we also offer ELISpot Development Modules, which provide a flexible, do-it-yourself format for ELISpot development.

• High sensitivity – ELISpot Assays can measure responses with frequencies below 1 in 100,000 cells
• No in vitro expansion of cells required
• High-throughput – ELISpot Assays use only a small number of primary cells

Learn more about ELISpot Assays.

Isolation Kits

Positive and negative cell selection kits to isolate immune cells for cell culture.

Animal-free Recombinant Proteins

Provide a seamless transition from preclinical research into clinical manufacturing, ensure defined culture conditions, and eliminate regulatory or ethical concerns associated with animal-derived reagents. Our Animal-free proteins are produced using identical expression systems and manufacturing methods as our Animal-free GMP-grade proteins.

GMP Proteins

Manufactured under guidelines that allow for their use as ancillary materials in cell therapy manufacturing processes. These proteins undergo extensive quality control testing and come with comprehensive documentation and full transparency and traceability of source and manufacturing system. Liquid, process-sized, and closed process options are also available for GMP cytokines. Learn more.

Cell Proliferation and Viability Assays

Reagents for studying cell proliferation, viability, and cytotoxicity including fluorescent reporter dyes and MTT assays.

Custom Services

If you can’t find the reagents you need on our website, contact us. Our custom services team will work with you to create the custom protein, antibody, ELISA, or Luminex assay that fits your research needs.