Elicit: CD28 Costimulation and Downstream Pathways
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, which cascades to strongly 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, with reduced IκB-α phosphorylation indicating impaired NF-κB regulation. 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 by 30-50%, and mitochondrial oxygen consumption.
Selective CD28 blockade while preserving CTLA-4 promotes regulatory pathways, upregulating Foxp3, PD-1, IDO, and CTLA-4 expression, which contribute to tolerance induction. However, when CD28 is eliminated, alternative costimulatory pathways including CD40-CD154, CD134-CD134L, and CD122 signaling can sustain T cell responses, particularly in memory CD8+ T cells and stringent rejection models. These pathway alterations translate into 36-73% inhibition of T cell proliferation, decreased type 1 cytokines (IL-2, IFNγ), increased regulatory cytokines (IL-10), and functional outcomes including donor-specific tolerance and prolonged allograft survival in transplantation models.
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: n = 10
Paper search
We performed a semantic search across over 138 million academic papers from the Elicit search engine, which includes all of Semantic Scholar and OpenAlex.
Search results: 200 total results from Elicit.
Screening
Criteria:
- CD28 Blockade Intervention: Does the study investigate CD28 costimulation blockade through any method?.
- Signaling Pathway Measurement: Does the study measure downstream signaling pathways, molecular cascades, or intracellular signaling events following CD28 blockade?
- Appropriate Controls: Does the study include appropriate control groups?
- Experimental Study Type: Is this an experimental study?
- Mechanistic Focus: Does the study include mechanistic signaling pathway data?
- Blockade vs Stimulation: Does the study examine CD28 blockade?
- Publication Type: Is this a full research article?
- Pathway Analysis Inclusion: Does the study include signaling pathway analysis?
Data extraction
We asked a large language model to extract data from each study including the following:
CD28 Blockade Method:
Extract details about how CD28 costimulation was blocked, including:
Specific agent used, Dosage/concentration, Timing of blockade, Route of administration, Any combinatorial treatments
Experimental System:
Extract the experimental model and context where CD28 blockade effects were studied.
Target Cell Types:
Extract which specific cell types were analyzed.
Signaling Pathways Analyzed:
Extract all downstream signaling pathways that were measured after CD28 blockade.
Pathway Changes Observed:
Extract the specific alterations in downstream signaling pathways caused by CD28 blockade.
Mechanistic Insights:
Extract any mechanistic explanations for how CD28 blockade leads to downstream signaling pathway changes.
Functional Consequences:
Extract functional outcomes that result from altered downstream signaling pathways after CD28 blockade.
Results
Characteristics of Included Studies
Ten studies examined downstream signaling pathway alterations following CD28 costimulation blockade.
| 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 (GVHD/rejection) | Human | Anti-B7.1/B7.2 mAbs, 72h | CD4+ T cells, monocytes |
| Koji Kishimoto et al., 2000 | Yes | Cardiac transplant | Transplantation | Mouse (BALB/c, C57BL/6) | 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 (CD4−/−CD28−/−, CD8−/−CD28−/−) | 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 1 µg/ml | 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 (C57BL/6J→CBA/J) | CTLA4Ig + anti-CD2 mAb at transplant | T lymphocytes (CD4+, CD8+) |
| David V. Mathews et al., 2018 | No | Transplant | Transplantation (kidney) | Mouse, NHP | Belatacept | CD8+ memory T cells |
| L. Tuosto & O. Acuto, 1998 | No | In vitro T cells, ex vivo TCR-Tg | Normal activation | Human (Jurkat), Mouse | CD28/B7 blockade | Jurkat T cells, primary T cells |
| T. Zhang et al., 2011 | Yes | Cardiac transplant | Transplantation | Mouse (C57BL/6, BALB/c) | α28scFv 100 µg bid ip, 2 wks | CD4+ T cells, regulatory T cells |
Proximal TCR Signaling Pathways
CD28 blockade profoundly impaired the earliest TCR signaling events. When CD28/B7 interaction was inhibited, TCR-induced tyrosine phosphorylation of the ζ chain and ZAP-70 was significantly reduced. This proximal signaling defect cascaded to downstream pathways.
| Signaling molecule | Direction of change | Cell type | Study |
|---|---|---|---|
| ζ chain phosphorylation | Downregulated | Jurkat T cells, primary mouse T cells | Tuosto & Acuto, 1998 |
| ZAP-70 phosphorylation | Downregulated | Jurkat T cells, primary mouse T cells | Tuosto & Acuto, 1998 |
| Ca2+/calcineurin pathway | Downregulated | Jurkat T cells | Tuosto & Acuto, 1998 |
| ERK/MAPK pathway | Downregulated | Jurkat T cells | Tuosto & Acuto, 1998 |
| JNK pathway | Downregulated | Jurkat T cells | Tuosto & Acuto, 1998 |
Transcription Factor Activation
CD28 blockade consistently suppressed critical transcription factors involved in T cell activation.
| Transcription factor | Direction of change | Magnitude | Cell type | Study |
|---|---|---|---|---|
| NF-κB | Downregulated | Not quantified | CD4+/CTLA-4+ T cells | Olsson et al., 1999 |
| AP-1 | Downregulated | Not quantified | CD4+/CTLA-4+ T cells | Olsson et al., 1999 |
| IκB-α phosphorylation | Reduced | Not quantified | CD4+/CTLA-4+ T cells | Olsson et al., 1999 |
| NFκB | Downregulated | Pronounced | CD4+ T cells | G. Gorgun et al., 2007 |
| NFκB | Downregulated | Not quantified | CD4+ T cells | G. Gorgun et al., 2008 |
IL-27 Signaling Pathway
IL-27, a heterodimer of p28 and EBI3 produced by antigen-presenting cells, regulates adaptive immunity by controlling T cell proliferation, Th1 differentiation, and IFNγ synthesis.
| Component | Direction of change | Cell type | Measurement | Study |
|---|---|---|---|---|
| p28 | Decreased | Monocytes | Gene and protein expression | G. Gorgun et al., 2007 |
| EBI3 | Decreased | Monocytes | Gene and protein expression | G. Gorgun et al., 2007 |
| IL-27R | Decreased | CD4+ T cells | Gene expression | G. Gorgun et al., 2008 |
| STAT3 | Decreased | CD4+ T cells | Gene expression | G. Gorgun et al., 2007, 2008 |
| pSTAT1 | Downregulated | CD4+ T cells | Gene and protein expression | G. Gorgun et al., 2007, 2008 |
Metabolic Signaling Pathways
CD28 blockade markedly disrupted T cell metabolic fitness.
| Metabolic component | Direction of change | Magnitude | Measurement method | Study |
|---|---|---|---|---|
| AKT phosphorylation | Reduced | Not quantified | Flow cytometry | Zhang et al., 2019 |
| mTOR activity | Reduced | Not quantified | Flow cytometry | Zhang et al., 2019 |
| Glut1 expression | Downregulated | Not quantified | Gene expression | Zhang et al., 2019 |
| Glycolytic enzymes (HK2, PFK1, GAPDH, LDH) | Downregulated | 30-50% reduction | Gene expression | Zhang et al., 2019 |
| HIF1α | Reduced | Not quantified | Flow cytometry | Zhang et al., 2019 |
| Mitochondrial oxygen consumption | Diminished | Not quantified | Seahorse analysis | Zhang et al., 2019 |
Regulatory and Tolerance-Associated Pathways
CD28 blockade while preserving CTLA-4 signaling promoted regulatory pathways and tolerance mechanisms.
| Regulatory molecule | Direction of change | Measurement | Functional association | Study |
|---|---|---|---|---|
| Foxp3 | Upregulated | Real-time RT-PCR | Regulatory T cell marker | T. Zhang et al., 2011 |
| CTLA-4 | Upregulated | Real-time RT-PCR | Inhibitory receptor | T. Zhang et al., 2011 |
| PD-1 | Upregulated | Real-time RT-PCR | Inhibitory receptor | T. Zhang et al., 2011 |
| FasL | Upregulated | Real-time RT-PCR | Apoptosis mediator | T. Zhang et al., 2011 |
| IDO | Upregulated (mRNA enriched) | Real-time RT-PCR | Immunoregulatory enzyme | T. Zhang et al., 2011 |
Alternative Costimulatory Pathways
When CD28 signaling was eliminated, compensatory costimulatory pathways that could drive T cell responses were identified.
| Alternative pathway | Evidence for compensation | Cell type | Study |
|---|---|---|---|
| CD40-CD154 | Prolonged survival when blocked in CD28−/− mice | CD4+ and CD8+ T cells | Habicht et al., 2007 |
| CD134-CD134L | Prolonged survival when blocked in CD28−/− mice | CD4+ and CD8+ T cells | Habicht et al., 2007 |
| CD122 (IL-15R) | Critical for costimulation-independent responses | Memory CD8+ T cells | Mathews et al., 2018 |
Functional Consequences Across Studies
The downstream signaling alterations translated into consistent functional outcomes across experimental systems. T cell proliferation was suppressed in all studies that measured it, ranging from 36-73% inhibition. Cytokine production patterns shifted toward a less inflammatory profile, with decreased type 1 cytokines (IL-2, IFNγ, IL-15) and increased regulatory cytokines (IL-10).
In transplantation models, CD28 blockade induced donor-specific tolerance, prolonged allograft survival, prevented chronic rejection, and reduced alloantibody production. The vasculitis model demonstrated reduced tissue-infiltrating T cells, suppressed vasculitis, and prevention of vessel wall remodeling.
Synthesis
The studies reveal a coordinated cascade of signaling disruptions initiated by CD28 blockade. Notably, CD28 blockade does not simply remove a costimulatory signal but actively remodels the signaling landscape. The metabolic dimension—glucose transporter expression, glycolytic enzyme activity, and mitochondrial function—represents a previously underappreciated mechanism of CD28 action.
The heterogeneity in experimental systems provides complementary insights. In vitro systems enabled precise dissection of early signaling events, while ex vivo MLRs revealed effects on multiple cell types including bystander populations. Animal models demonstrated sustained effects on immune responses and tissue outcomes.
Some apparent contradictions illuminate context-dependent effects. CD28 blockade consistently induces tolerance in most transplant models but can fail in stringent models like skin transplantation. This discrepancy is explained by alternative costimulatory pathways that can sustain rejection in CD28’s absence.
The temporal dynamics of pathway alterations also merit attention. Metabolic effects occur within 30 minutes, transcription factor suppression is evident within hours, proliferative suppression manifests at 72 hours, and regulatory pathway upregulation persists for at least 100 days.
Human versus mouse studies showed largely concordant results for shared pathways. Both species demonstrated NF-κB suppression, impaired proximal TCR signaling, and reduced T cell proliferation. The IL-27 pathway was examined only in human cells, while most regulatory pathway analyses occurred in mice.