In the adaptive immune response, T cell functional activation relies on a critical two-signal regulatory mechanism. T cell receptor (TCR) recognition of the MHC-antigen peptide complex on the surface of antigen-presenting cells (APCs) provides the first signal. The co-stimulatory receptor CD28 binding to its ligands B7.1 (CD80) and B7.2 (CD86) on APCs provides the essential second signal. Together, these signals drive full T cell activation, clonal proliferation, and effector differentiation. The CD28 signal drives T cell clonal expansion, effector differentiation (e.g., IL-2 secretion), and metabolic reprogramming. Its precise regulation has become a central target in tumor immunotherapy and autoimmune disease intervention.
 

Target Introduction
 

CD28 is a type I transmembrane protein constitutively expressed on the surface of most CD4+ and CD8+ T cells (expression declines with age), existing as a homodimer. Its extracellular region contains an immunoglobulin variable-like domain, which specifically binds its ligands CD80/CD86 via a conserved MYPPPY motif. The human CD28gene contains 4 exons, encoding 220 amino acids. The mature protein is a glycosylated dimer with a molecular weight of approximately 44 kDa. It is not only an amplifier of TCR signals but also initiates a unique intracellular signaling network, systematically programming T cell effector function and immune memory from transcriptional regulation to epigenetic modification.

Image source: CD28 co-stimulation: novel insights and applications in cancer immunotherapy
 

Core Functions
 

1) Signaling Transduction Network
 

CD28 activation depends on its intracellular tail motifs, YMNM and PYAP, which recruit and activate distinct downstream pathways:
 

YMNM Motif:​ Primarily recruits PI3K, activating the Akt-mTOR pathway, promoting cell metabolism and survival.
 

PYAP Motif:​ Phosphorylated by kinases like LCK, activates PKCθ, leading to the activation of key transcription factors NF-κB, AP-1, and NFAT, driving effector gene expression.
 

Adaptor Protein Activation:​ Via proteins like GRB2/GADS, activates the Ras-MAPK pathway, promoting cell proliferation.
 

2) Dual Regulation of Immunity and Metabolism
 

Regulating Immune Balance:​ The CD28 signal has duality. On one hand, it is essential for the activation and proliferation of effector T cells. On the other hand, it is equally indispensable for the thymic development, peripheral homeostasis, and suppressive function of regulatory T cells (Tregs). Disruption of this balance is associated with various autoimmune diseases.
 

Driving Metabolic Reprogramming:​ CD28 is a central hub connecting immune signals and cellular metabolism. By upregulating the glucose transporter GLUT1, it strongly enhances glycolysis to fuel activated T cells. Simultaneously, it can reshape mitochondrial metabolism (e.g., enhancing fatty acid oxidation via CPT1A), influencing memory T cell formation and delaying the functional exhaustion process of CD8+ T cells, thereby determining their differentiation fate and maintenance of effector functions.
 

Image source: CD28 co-stimulation: novel insights and applications in cancer immunotherapy
 

Association with Core Diseases
 

1) Rheumatoid Arthritis (RA)
 

In RA pathogenesis, autoreactive T cells help B cells produce high-affinity autoantibodies and secrete pro-inflammatory cytokines, exacerbating synovial inflammation and bone destruction. The co-stimulatory signal mediated by CD28 is a core link driving the activation of these autoreactive T cells. The PI3K-Akt pathway activated via the YMNM motif and the PKCθ-NF-κB pathway activated via the PYAP/Lck motif together promote T cell proliferation, survival, and production of pro-inflammatory factors (e.g., TNF-α, IL-6, IL-17).
 

2) Multiple Sclerosis (MS)
 

Autoreactive CD4+ T cells (particularly Th1 and Th17 subsets), once activated, cross the blood-brain barrier and attack central nervous system myelin, leading to demyelination. The CD28 signal is a key driver for the initial activation and expansion of these pathogenic T cells in peripheral lymph nodes. This signal drives the differentiation and function of Th17 and Th1 cells by promoting metabolic reprogramming (e.g., enhancing glycolysis) and stabilizing the expression of pro-inflammatory transcription factors (e.g., RORγt, T-bet).
 

3) Systemic Lupus Erythematosus (SLE)
 

Dysregulation of the CD28 signaling pathway exacerbates widespread immune system dysfunction. On one hand, excessive CD28 co-stimulation promotes the abnormal activation of autoreactive T follicular helper (Tfh) cells, which in turn help B cells produce large quantities of pathogenic autoantibodies (e.g., against dsDNA). On the other hand, the CD28 signal is crucial for maintaining the function and homeostasis of regulatory T cells. In SLE patients, an imbalance in CD28 signaling may exist: effector T cells are overactivated, while Treg function may be impaired. This imbalance is associated with abnormal activation of signaling pathways like PI3K-mTOR, leading to a cytokine storm and breakdown of immune tolerance.
 

4) Type 1 Diabetes (T1D)
 

In T1D, CD28 signaling participates in the autoimmune attack against pancreatic islet β-cells. The activation and expansion of islet-specific autoreactive CD8+ and CD4+ T cells depend on the co-stimulatory signal provided by CD28. This signaling pathway, particularly through NF-κB activation, promotes effector T cell infiltration of the islets and destruction of β-cells. Meanwhile, CD28 signaling also regulates the development and function of Tregs. Studies have found that immunotherapies like anti-CD3 antibodies can induce the generation of suppressive CD8+ T cells, which partly depends on modulation of T cell co-stimulatory pathways.
 

Mouse Models
 

CD28 KO Mice:​ Complete knockout of the CD28gene. They exhibit impaired T cell activation and abnormal antibody class switching; Treg numbers are significantly reduced. Primarily used to study the role of CD28 in autoimmune disease pathogenesis.
 

CD28 Humanized Mice:​ The extracellular domain of the mouse Cd28gene is replaced with the human sequence, leading to the expression of human CD28 protein on T cell surfaces while maintaining normal T cell development and homeostasis. Primarily used for the in vivo evaluation of the efficacy and safety of CD28-targeting antibody drugs.
 

hCD3/hCD28 Double Humanized Mice:​ Both CD3and CD28genes are humanized, achieving humanization of both T cell receptor signaling and co-stimulatory signaling. Primarily used in the preclinical development of T-cell engager (TCE) 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. The 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 CD28-related mouse models according to client needs, such as CD28 KO mice, CD28 humanized mice, and hCD3/hCD28 double humanized mice. Inquiries are welcome.
 

References:

1.Lotze MT, Olejniczak SH, Skokos D. CD28 co-stimulation: novel insights and applications in cancer immunotherapy. Nat Rev Immunol. 2024 Dec;24(12):878-895. doi: 10.1038/s41577-024-01061-1. Epub 2024 Jul 25. PMID: 39054343; PMCID: PMC11598642.

2.Esensten JH, Helou YA, Chopra G, Weiss A, Bluestone JA. CD28 Costimulation: From Mechanism to Therapy. Immunity. 2016 May 17;44(5):973-88. doi: 10.1016/j.immuni.2016.04.020. PMID: 27192564; PMCID: PMC4932896.

3.Porciello N, Kunkl M, Tuosto L. CD28 between tolerance and autoimmunity: the side effects of animal models. F1000Res. 2018 May 30;7:F1000 Faculty Rev-682. doi: 10.12688/f1000research.14046.1. PMID: 29904580; PMCID: PMC5981186.

4.Ceeraz S, Thompson CR, Beatson R, Choy EH. Harnessing CD8+CD28- Regulatory T Cells as a Tool to Treat Autoimmune Disease. Cells. 2021 Nov 1;10(11):2973. doi: 10.3390/cells10112973. PMID: 34831195; PMCID: PMC8616472.