# CD28 Costimulation and Downstream Pathways

## What downstream signaling pathways are altered when CD28 costimulation is blocked?

CD28 costimulation blockade disrupts proximal TCR signaling (ζ chain, ZAP-70, Ca2+/calcineurin, ERK/MAPK, JNK), suppresses transcription factors (NF-κB, AP-1), reduces IL-27 pathway signaling (STAT3, pSTAT1), impairs metabolic pathways (AKT-mTOR-HIF-1α axis, glucose uptake, glycolysis), and when selective, upregulates regulatory pathways (Foxp3, PD-1, IDO, CTLA-4).

# Abstract

CD28 costimulation blockade disrupts multiple downstream signaling pathways across a coordinated cascade. At the proximal level, CD28 blockade impairs TCR-induced tyrosine phosphorylation of the ζ chain and ZAP-70, leading to diminished Ca2+/calcineurin, ERK/MAPK, and JNK pathway activation. Transcription factors critical for T cell activation, particularly NF-κB and AP-1, are potently suppressed. CD28:B7 signaling is required for monocyte IL-27 production, and blockade decreases IL-27 pathway components (p28, EBI3, IL-27R, STAT3, pSTAT1) in both monocytes and CD4+ T cells. Metabolically, CD28 blockade disrupts the AKT-mTOR-HIF-1α axis, reducing glucose transporter Glut1 expression, glycolytic enzyme levels, and mitochondrial oxygen consumption.

# Methods

We analyzed 10 sources from an initial pool of 200, using 8 screening criteria. Each paper was reviewed for 7 key aspects that mattered most to the research question.

### Records from Elicit search

- **n** = 200
  - Papers screened using: CD28 Blockade Intervention, Signaling Pathway Measurement, Appropriate Controls, Experimental Study Type, Mechanistic Focus, Blockade vs Stimulation, Publication Type, Pathway Analysis Inclusion
- **n** = 200 Papers screened out
- **n** = 190 Papers included for extraction

## Data extraction

### CD28 Blockade Method
- Specific agent used: Anti-CD28 antibody
- Dosage/concentration: 1 µg/ml
- Timing of blockade: Not specified
- Route of administration: Not specified

### Experimental System
- Model system: In vitro cell culture, animal model
- Disease context: Transplantation, autoimmunity
- Species: Human, mouse

### Target Cell Types
- Primary cell type studied: CD4+ T cells
- Cell activation state: Activated

### Signaling Pathways Analyzed
- Pathway names: NF-κB, AKT, mTOR

### Pathway Changes Observed
- Direction of change: Downregulated
- Magnitude of effect: 30-50% reduction in glycolytic enzymes

### Mechanistic Insights
- Proposed mechanism: CD28 blockade disrupts T-cell metabolic fitness by reducing glucose utilization.

### Functional Consequences
- T cell proliferation: Suppressed
- Cytokine production: Altered
- Therapeutic implications: Novel therapeutic opportunities for disease-modifying strategies.

## Results

### Characteristics of Included Studies
Ten studies examining downstream signaling pathway alterations following CD28 costimulation blockade.

#### Study Details
| Study | Full text retrieved? | Model system | Disease context | Species | CD28 blockade method | Target cell types |
|-------|---------------------|--------------|-----------------|---------|---------------------|------------------|
| G. Gorgun et al., 2007 | No | Ex vivo MLR | Transplantation | Human | Anti-B7.1/B7.2 mAbs, 72h | CD4+ T cells, monocytes |
| Koji Kishimoto et al., 2000 | Yes | Cardiac transplant | Transplantation | Mouse | CTLA4Ig 250 µg ip, d2 | T cells (Th1/Th2) |
| G. Gorgun et al., 2008 | No | Ex vivo MLR | Transplantation | Human | Anti-B7.1/B7.2 mAbs, 72h | CD4+ T cells, monocytes |
| A. Habicht et al., 2007 | No | Skin transplant | Transplantation | Mouse | CD28 knockout | CD4+ and CD8+ effector-memory T cells |
| Hui Zhang et al., 2019 | Yes | Human artery-NSG chimera | Vasculitis (GCA) | Human/Mouse | Anti-CD28dAb | CD4+ T cells, CD4+CD103+ TRM |
| C. Olsson et al., 1999 | No | In vitro blastoid T cells | Normal activation | Mouse | CTLA-4 mAb | CD4+/CTLA-4+ T cells |
| J. Woodward et al., 1996 | No | Cardiac transplant | Transplantation | Mouse | CTLA4Ig + anti-CD2 mAb | T lymphocytes (CD4+, CD8+) |
| David V. Mathews et al., 2018 | No | Transplant | Transplantation | Mouse | Belatacept | CD8+ memory T cells |
| L. Tuosto & O. Acuto, 1998 | No | In vitro T cells | Normal activation | Human (Jurkat), Mouse | CD28/B7 blockade | Jurkat T cells, primary T cells |
| T. Zhang et al., 2011 | Yes | Cardiac transplant | Transplantation | Mouse | α28scFv 100 µg bid | CD4+ T cells, regulatory T cells |

## Synthesis
The studies reveal a coordinated cascade of signaling disruptions initiated by CD28 blockade. The discovery that CD28:B7 signaling is required for monocyte IL-27 production reveals indirect effects on bystander cells beyond the directly targeted T cells. Metabolic effects occur rapidly, affecting T cell proliferation and differentiation.

## References
[1]: (G. Gorgun et al. 2007) Costimulatory Blockade during Mixed Lymphocyte Reaction Prevents IL-27 Upregulation and Signaling. Blood
[2]: (Koji Kishimoto et al. 2000) The role of CD154-CD40 versus CD28-B7 costimulatory pathways in regulating allogeneic Th1 and Th2 responses in vivo. Journal of Clinical Investigation
[3]: (Hui Zhang et al. 2019) CD28 Signaling Controls Metabolic Fitness of Pathogenic T Cells in Medium and Large Vessel Vasculitis. Journal of the American College of Cardiology