CTLA-4/CD28 Signaling Pathway

Figure 1. CTLA-4/CD28 signaling pathway.


Antigen-specific activation of naive CD4+ T cell is one of the important events in the initiation of adaptive immunity. Therefore, the event is controlled by a finely tuned balance of stimulatory and inhibitory regulatory signals. There have two distinct signals to regulate adaptive immunity. The first signal of this two-signal model is antigen-specific and is produced by interaction of the TCR with antigenic peptide presented in context with MHC antigens. The second signal is referred to the T cell surface molecule CD28. CD28 delivers a costimulatory signal upon interaction with B7 molecules present on APC. Activation of the TCR in the presence of costimulatory signals leads to T cell clonal expansion and initiation of effector functions. Due to the need to maintain a balance between immune activation and tolerance, there is also a protein molecule, cytotoxic T lymphocyte-associated molecule-4(CTLA-4), that antagonizes CD28. CTLA-4 is a cell surface molecule that is closely related to CD28, and it shows a potent negative regulator of T cell activation.

Pathway regulation

CD28 signaling

In addition to TCR engagement by peptide, ligation of co-stimulatory molecules is also required for a productive immune response to occur. CD28 is the best characterized and most effective co-stimulatory molecule expressed by naive and primed T cells. CD28 is a 44-kDa glycoprotein that binds to B7-1 (CD80) and B7-2 (CD86) on antigen-presenting cells (APCs) CD28 might be associate, in its unphosphorylated state, with the serine/threonine protein phosphatase 2A (PP2A). On T-cell stimulation, CD28 undergoes phosphorylation on its intracellular tyrosine residues (Y), presumably leading to dissociation from PP2A and recruitment of phosphatidylinositol 3-kinase (PI3K) and (growth factor-receptor-bound protein 2 (GRB2). Activation of PI3K, which induces phosphorylation of phosphatidylinositol (PI) into phosphatidylinositol 3-phosphate (PIP3), might promote activation of protein kinase B (PKB/Akt), followed by that of nuclear factor-κB (NF-κB), resulting in BCL-XL upregulation that favours T-cell survival. Akt activation might stimulate interleukin-2 (IL-2) production. PI3K is negatively regulated by phosphatase and tensin homologue (PTEN). The carboxy-terminal proline (P) -rich region might promote IL-2 production and proliferation, perhaps by recruiting and activating Lck.

CTLA-4 signaling

In addition to positive co-stimulatory molecules, inhibitory receptors have also been identified on T cells, such as the cytoplasmic tail of cytotoxic T-lymphocyte antigen 4(CTLA-4). CTLA-4 is expressed on activated T cells, is about 30% homologous with CD28 and binds to the same ligands as CD28. CD28 has an important residue (YMNM) that is critical for its effective signaling. The YMNM motif beginning at tyrosine 170 in particular is critical for the recruitment of SH2-domain containing proteins, especially PI3K, Grb2 and Gads. Similar to CD28, CTLA-4 contains also two tyrosine residues (Y) at positions 201 and 208 and a proline (P) -rich region. Y201 is packed within a YVKM motif. In its unphosphorylated form this motif allows association of CTLA-4 with AP50. This interaction results in clathrin-dependent endocytosis of CTLA-4, therefore limiting CTLA-4 surface expression. CTLA-4 might also bind with the serine/threonine protein phosphatase 2A (PP2A), although the functional consequence of this is not yet known. After TCR stimulation, CTLA-4 might undergo tyrosine phosphorylation by SRC kinases, inducing surface retention. Whether Y201  requires phosphorylation for CTLA-4 to associate with phosphatidylinositol 3-kinase (PI3K) and SH2-domain containing protein tyrosine phosphatase (SHP-2) remains controversial. Crosslinking of CTLA-4 has been found to reduce T-cell receptor (TCR)-dependent activation of the mitogen-activated protein (MAP) kinases ERK (extracellular signal-regulated kinase) and JNK (Jun N-terminal kinase), as well as of the transcription factors nuclear factor-κB (NF-κB), AP-1 and NF-AT (nuclear factor of activated T cells), and ligation of CTLA-4 during TCR stimulation results in decreased cytokine production by T cells and cell-cycle arrest. Indirect association of CTLA-4 with SHP-2 might result in dephosphorylation of the CD3 complex and subsequent inactivation of TCR-dependent signaling pathways. Alternatively, CTLA-4 might inhibit these T-cell functions by an as yet unidentified signaling pathway.

Cancer immunotherapy

Until now, treatment of tumors and drug development are still mainly focused on the idea of directly targeting cancer cells and killing tumor cells—such as surgery, radiotherapy, chemotherapy, and kinase inhibitors. However, for more than a century, people have also recognized the interaction of the immune system with cancer. Scientific research demonstrates that the immune system can identify and reject cancer cells as foreign, and tumors can use immunological checkpoints to protect themselves from immunological functions by abnormally expressing ligands that normally interact with inhibitory immune receptors. As the first immune checkpoint receptor discovered and identified, cytotoxic T-lymphocyte antigen 4 (CTLA-4), it shows a close relationship with cancer treatment. In animal models, antibodies that block CTLA-4 can mediate the regression of established tumors. This observation directly led to the clinical start of detection of CTLA-4 monoclonal antibodies in cancer patients. In 2011, the FDA approved the first immune checkpoint blocker, iprocidox, for advanced melanoma. However, many of the adverse reactions in patients treated with anti-CTLA-4 such as rash, colitis, thyroiditis, and hepatitis are similar to autoimmune diseases and are consistent with the mechanism of checkpoint blockade. Although CTLA-4 immunotherapy still has some deficiencies, it provides us with new ways to treat tumors, and giving us new tools to treat cancer.


1. Maria L A., et al. T-CELL REGULATION BY CD28 AND CTLA-4. Nature, 2001, 1: 220-228.
2. Rebecca L., et al. Challenges and opportunities in targeting the CD28/CTLA-4 pathway in transplantation and autoimmunity. Expert Opinion on Biological Therapy, 2017, 17(8): 1001-1012.
3. Kathy D M., Graham L G. The role of CTLA-4 in the regulation of T cell immune responses. Immunology and Cell Biology, 1999, 77: 1-10.

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