As the first immune checkpoint molecule approved globally by the U.S. Food and Drug Administration (FDA)​ for cancer immunotherapy, CTLA-4 (Cytotoxic T-Lymphocyte-Associated Antigen 4, CD152) has been a star target in the field of tumor immunoregulation since its cloning in the 1990s. The approval of its antibody drug, Ipilimumab (Yervoy®), marked the beginning of the immune checkpoint blockade era, and it remains widely used for treating refractory tumors such as melanoma and renal cell carcinoma.
 

Target Introduction
 

Cytotoxic T-Lymphocyte-Associated Antigen 4 (CTLA-4), also known as CD152, is an important member of the immunoglobulin superfamily, sharing high homology with the co-stimulatory molecule receptor CD28 on the T cell surface. From a molecular structure perspective, CTLA-4 is a type I transmembrane protein receptor, mainly expressed on the surface of activated CD4+ and CD8+ T cells. It is also constitutively highly expressed on regulatory T cells (Tregs), serving as a key molecule in maintaining immune tolerance and preventing autoimmune responses.
 

Core Functions: Triple-Mechanism Negative Regulation of T Cell Immunity
 

1.Competitive Blockade of B7 Ligands, Inhibiting Co-stimulatory Signal Transmission:
 

CTLA-4 competitively binds to the same B7 ligands (CD80/CD86) as CD28, and CTLA-4's affinity for these ligands is significantly higher than that of CD28. Through its high-affinity IgV domain, CTLA-4 preferentially occupies B7 molecules on the surface of Antigen-Presenting Cells (APCs), blocking CD28 from receiving the co-stimulatory signal. This directly weakens the second signal required for T cell activation. This leads to insufficient activation of the downstream PI3K/AKT and NF-κB pathways, thereby inhibiting IL-2 production, cell cycle progression (G1 phase arrest), and clonal expansion, constituting the primary negative regulatory mechanism for early T cell activation.
 

2.Intracellular Recruitment of Phosphatases, Interfering with Positive Signal Transduction Pathways:
 

The intracellular short tail of CTLA-4 contains tyrosine phosphorylation sites that specifically recruit phosphatases SHP-2 and PP2A. SHP-2 dephosphorylates key nodes in the TCR signaling pathway (e.g., ZAP-70, LAT), blocking the PLCγ1-Ca²⁺/PKCθ signal cascade. PP2A directly dephosphorylates AKT, inhibiting the PI3K/AKT/mTOR pathway. These effects collectively weaken the key signals required for T cell activation, metabolic reprogramming (e.g., glucose uptake), and effector gene (e.g., IFN-γ) expression, reinforcing immune suppression at the intracellular level.
 

3.Mediating B7 Ligand Endocytosis and Degradation, Depleting the Source of Co-stimulation:
 

In regulatory T cells that constitutively express CTLA-4, the CTLA-4-B7 complex can be internalized into the cell via clathrin-dependent endocytosis. The B7 ligands are subsequently transported to lysosomes for degradation. This process can sustainably reduce the density of B7 molecules on the surface of APCs, physically depleting the available ligands for CD28 in the microenvironment, thereby creating a local immunosuppressive environment. This mechanism is core to the "bystander suppression" function of Tregs, enabling them to broadly inhibit the activation of neighboring effector T cells.

 

Image source: Mechanisms of immune checkpoint inhibitors: insights into the regulation of circular RNAs involved in cancer hallmarks

 

Association with Core Diseases: From Autoimmunity to Tumor Immunity
 

1.Autoimmune Diseases:
 

CTLA-4 is a key negative regulatory molecule for maintaining immune system tolerance. Its functional loss or defect leads to the failure of this immune checkpoint for T cell activation, causing sustained enhancement of CD28-mediated co-stimulatory signals. This triggers abnormal activation of the downstream PI3K/AKT and NF-κB pathways, promoting excessive proliferation of T cells, their differentiation into effector cells, and the massive release of pro-inflammatory cytokines. These attack multiple tissues and organs such as the skin, intestines, and liver, thereby triggering severe systemic autoimmune diseases.
 

2.Tumor Immunity:
 

In the tumor microenvironment, tumor cells can utilize the CTLA-4 pathway to mediate immune escape through various mechanisms. These include upregulating the expression of B7 ligands on the surface of APCs or recruiting regulatory T cells that highly express CTLA-4 to infiltrate the tumor site, thereby inhibiting the activation, proliferation, and killing functions of effector T cells. Anti-CTLA-4 monoclonal antibodies (e.g., Ipilimumab) can specifically block the binding of CTLA-4 to B7 ligands, restore CD28-dependent co-stimulatory signals, relieve the functional inhibition of T cells, and promote their differentiation into cytotoxic T lymphocytes with tumor-killing activity, thereby enhancing anti-tumor immune responses.
 

Image source: CTLA-4 and PD-1 Pathways
 

Mouse Models
 

CTLA-4 KO Mice:​ Complete knockout of the CTLA-4gene. They exhibit T cell over-activation, multi-organ autoimmune damage, and early death. This is a core model for studying the role of CTLA-4 in immune tolerance.
 

CTLA-4 Conditional Knockout Mice:​ Knockout of CTLA-4in specific T cell subsets (e.g., Tregs or effector T cells), allowing precise study of its functional mechanisms in different cell types.
 

Humanized CTLA-4 Mice:​ Express human CTLA-4, used for evaluating the efficacy and safety of humanized antibody drugs.
 

Supporting Mechanism Research and Drug Development
 

Gene therapy offers hope for common diseases, but its development and validation rely heavily on animal model support. MingCeler Biotech, leveraging its self-developed TurboMice™ technology, has developed multiple disease mouse models. TurboMice™ technology overcomes the technical challenges of long mouse model generation cycles and low success rates for complex models, enabling editing at almost any target gene locus. Complete homozygous gene-edited mouse models can be prepared directly from embryonic stem cells in as little as two months.
 

MingCeler Biotech​ can customize various CTLA-4-related mouse models according to client needs, such as CTLA-4 KO mice, CTLA-4 conditional knockout mice, and humanized CTLA-4 mice. Inquiries are welcome.
 

References:

1.Buchbinder EI, Desai A. CTLA-4 and PD-1 Pathways: Similarities, Differences, and Implications of Their Inhibition. Am J Clin Oncol. 2016 Feb;39(1):98-106. doi: 10.1097/COC.0000000000000239. PMID: 26558876; PMCID: PMC4892769.

2.Chen H, Yang H, Guo L, Sun Q. The Role of Immune Checkpoint Inhibitors in Cancer Therapy: Mechanism and Therapeutic Advances. MedComm (2020). 2025 Oct 5;6(10):e70412. doi: 10.1002/mco2.70412. PMID: 41059489; PMCID: PMC12497686.