# 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](https://www.semanticscholar.org/) and [OpenAlex](https://openalex.org/).
We ran this query: "What downstream signaling pathways are altered when CD28 costimulation is blocked?"
The search returned 200 total results from Elicit.
We retrieved 200 papers most relevant to the query for screening.

## Screening

We screened in sources based on their abstracts that met these criteria:
- **CD28 Blockade Intervention**: Does the study investigate CD28 costimulation blockade through any method (antibodies, genetic knockout, pharmacological inhibitors, or other blocking agents)?
- **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 (untreated, vehicle-treated, or isotype controls)?
- **Experimental Study Type**: Is this an in vitro, ex vivo, or in vivo experimental study?
- **Mechanistic Focus**: Does the study include mechanistic signaling pathway data (not solely clinical outcomes)?
- **Blockade vs Stimulation**: Does the study examine CD28 blockade (not solely CD28 agonism or stimulation)?
- **Publication Type**: Is this a full research article (not a conference abstract, case report, or editorial)?
- **Pathway Analysis Inclusion**: Does the study include signaling pathway analysis (not only phenotypic or functional outcomes)?  
We considered all screening questions together and made a holistic judgement about whether to screen in each paper.

## Data extraction

We asked a large language model to extract each data column below from each paper. We gave the model the extraction instructions shown below for each column.
- **CD28 Blockade Method**:

Extract details about how CD28 costimulation was blocked, including:
- Specific agent used (e.g., anti-CD28 antibody, selective CD28 blockers, CTLA-4 ligands)
- Dosage/concentration
- Timing of blockade (when initiated, duration)
- Route of administration
- Any combinatorial treatments

- **Experimental System**:

Extract the experimental model and context where CD28 blockade effects on signaling pathways were studied, including:
- Model system (in vitro cell culture, animal model, ex vivo assays)
- Disease/condition context (transplantation, autoimmunity, normal activation, etc.)
- Species (human, mouse, etc.)
- Specific experimental setup relevant to pathway analysis

- **Target Cell Types**:

Extract which specific cell types were analyzed for downstream signaling pathway changes after CD28 blockade, including:
- Primary cell type studied (CD4+ T cells, CD8+ T cells, monocytes, etc.)
- Cell activation state (naive, activated, memory, etc.)
- Any cell subsets specifically examined
- Tissue location if relevant (blood, lymphoid organs, transplanted tissue, etc.)

- **Signaling Pathways Analyzed**:

Extract all downstream signaling pathways that were measured after CD28 blockade, including:
- Specific pathway names (NF-κB, AP-1, AKT, mTOR, metabolic pathways, etc.)
- Individual signaling molecules/proteins analyzed
- Measurement methods (Western blot, flow cytometry, gene expression, etc.)
- Time points of analysis
- Any pathway interactions or crosstalk studied

- **Pathway Changes Observed**:

Extract the specific alterations in downstream signaling pathways caused by CD28 blockade, including:
- Direction of change (upregulated, downregulated, no change)
- Magnitude of effect (fold-change, percentage change, statistical significance)
- Kinetics of changes (immediate, delayed, sustained, transient)
- Comparison to appropriate controls
- Any dose-response relationships

- **Mechanistic Insights**:

Extract any mechanistic explanations provided for how CD28 blockade leads to downstream signaling pathway changes, including:
- Proposed mechanism of action
- Key intermediary molecules or steps
- Relationship between CD28 and other costimulatory/inhibitory pathways
- Any evidence for direct vs. indirect effects
- Regulatory loops or feedback mechanisms identified

- **Functional Consequences**:

Extract functional outcomes that result from the altered downstream signaling pathways after CD28 blockade, including:
- Changes in T cell proliferation, differentiation, or survival
- Cytokine production alterations
- Metabolic changes
- Effects on immune responses (tolerance, rejection, etc.)
- Any therapeutic implications mentioned

## Results

## Characteristics of Included Studies

Ten studies examined downstream signaling pathway alterations following CD28 costimulation blockade. Three studies used human cells or tissues, one used both human and mouse cells, and six used murine systems exclusively. The majority focused on transplantation contexts, with one examining vasculitis and two studying normal T cell activation.

| 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, with strongly diminished tyrosine phosphorylation of Ca2+/calcineurin, ERK/MAPK, and JNK pathways. These findings suggest CD28 enhances TCR signaling capacity during the earliest stages of T cell-APC interaction.

| 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 |

The impairment of these proximal pathways provides a mechanistic explanation for how CD28 blockade disrupts T cell activation at its foundation, affecting all subsequent signaling cascades.

## Transcription Factor Activation

CD28 blockade consistently suppressed critical transcription factors involved in T cell activation. NF-κB and AP-1, both induced by CD3/CD28 stimulation, were potently suppressed by CTLA-4 ligation. The composition of NF-κB and AP-1 family members remained similar regardless of stimulation conditions, but their overall activity was reduced. Analysis of the NF-κB regulator IκB-α revealed reduced phosphorylation in CTLA-4 coengaged T cells compared to CD3/CD28 stimulation alone, suggesting the major impact of CTLA-4 ligation is inhibition of CD28-mediated signals rather than TCR-mediated signals.

Multiple studies documented NFκB suppression. In ex vivo mixed lymphocyte reactions with CD28:B7 blockade, CD4 T cells exhibited inactivation of NFκB at both transcriptional and translational levels. Similarly, CD28 blockade in the vasculitis model reduced NFκB activity.

| 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 |

The convergence of multiple studies on NF-κB suppression highlights this as a central mechanism through which CD28 blockade inhibits T cell activation, given NF-κB’s critical role in driving inflammatory gene expression.

## IL-27 Signaling Pathway

Two complementary studies from the same research group identified IL-27 signaling as a previously unrecognized target of CD28 costimulation blockade. 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.

In monocytes isolated from blocked MLRs, both p28 and EBI3 gene and protein expression levels were decreased. The effects extended to CD4 T cells, where IL-27R expression was reduced along with downstream signaling molecules including STAT3, pSTAT1, and NFκB. These changes were associated with decreased expression of type 1 cytokines (IL-2, IFNγ, IL-15) and increased expression of type 2 cytokines (IL-10), supporting a negative effect on Th1 differentiation.

| 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 |

The mechanism involves CD28:B7 signaling being required for monocyte production of IL-27, with decreased IL-27 and its downstream signaling molecules contributing to CD4 T alloantigen anergy induction by suppressing effector cytokines and Th1 cell differentiation. This represents a previously unrecognized crosstalk between CD28 and IL-27 pathways.

## Metabolic Signaling Pathways

CD28 blockade markedly disrupted T cell metabolic fitness, as comprehensively demonstrated in the vasculitis model. Blocking CD28 implicated this pathway in activating AKT signaling, which drives T-cell proliferation and differentiation of IFN-γ and IL-21-producing effector T cells.

The metabolic effects were profound and multifaceted. Expression of the glucose transporter Glut1 was highly sensitive to CD28 blockade, with 30-50% reduction in glycolytic enzyme expression (HK2, PFK1, GAPDH, LDH). Mitochondrial oxygen consumption was diminished, indicating broad metabolic impairment beyond glycolysis alone. The CD28-AKT-mTOR-HIF-1α pathway emerged as a critical regulatory axis, with CD28 stimulation providing a metabolic signal required for pathogenic effector functions.

| 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 |

Functionally, blocking CD28 was immunosuppressive by disrupting T-cell metabolic fitness, specifically the ability to utilize glucose. This metabolic suppression occurred within 30 minutes of stimulation, indicating rapid signaling effects. The metabolic defect impaired T-cell proliferation, differentiation into IFN-γ and IL-21-producing effector cells, and maintenance of tissue-resident memory T cells.

## Regulatory and Tolerance-Associated Pathways

CD28 blockade while preserving CTLA-4 signaling promoted regulatory pathways and tolerance mechanisms. In the α28scFv study, selective CD28 blockade for two weeks after cardiac transplantation was associated with increased proportion of early graft infiltration by regulatory T cells and increased expression of regulatory dendritic cell genes. Specifically, expression of Foxp3, CTLA-4, FasL, PD-1, and IDO was upregulated. This occurred at both early (days 10-12) and late (day 100) time points, indicating sustained effects.

| 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 |

Critically, blockade of CTLA-4 during α28scFv-based treatments led to prompt rejection and inhibited expression of Foxp3, PD-1, and IDO in the graft. This demonstrates that selective CD28 inhibition while sparing CTLA-4 promotes immunomodulatory mechanisms through at least two pathways: preventing maturation of pathogenic effectors while preserving CTLA-4-dependent immune regulation.

## Alternative Costimulatory Pathways

When CD28 signaling was eliminated, several studies identified compensatory costimulatory pathways that could drive T cell responses. In CD28-deficient mice, allograft rejection proceeded through CD40-CD154 and CD134-CD134L pathways. Blockade of both pathways significantly prolonged allograft survival in CD8−/−CD28−/− recipients and to a lesser extent in CD4−/−CD28−/− recipients. This prolongation was associated with reduced effector-memory T-cell generation, decreased Th1 cytokine production, and diminished T-cell proliferation.

Similarly, CD122 signaling through the IL-2 and IL-15 receptor β-chain emerged as critical for costimulation-independent memory CD8+ T cell responses. Combined costimulatory and CD122 blockade controlled proliferation and effector function of CD8+ T cells, with signaling through CD122 as a component of the high-affinity IL-15 receptor being critical for costimulation-independent memory CD8+ T cell recall.

| 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 |

These findings indicate that while CD28 is a dominant costimulatory pathway, multiple alternative routes can sustain T cell responses in its absence, particularly in memory populations and in stringent rejection models.

## 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, establishing therapeutic potential beyond transplantation.

## Synthesis

The studies reveal a coordinated cascade of signaling disruptions initiated by CD28 blockade. At the proximal level, CD28 enhances TCR signaling through ζ chain and ZAP-70 phosphorylation, which when blocked, impairs all downstream pathways including Ca2+/calcineurin, ERK/MAPK, and JNK. This proximal defect propagates to transcription factor suppression (NF-κB, AP-1), cytokine signaling alterations (IL-27 pathway), and metabolic reprogramming (AKT/mTOR/glycolysis).

Several mechanistic themes emerge. First, CD28 blockade does not simply remove a costimulatory signal but actively remodels the signaling landscape. 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. Second, the metabolic dimension—glucose transporter expression, glycolytic enzyme activity, and mitochondrial function—represents a previously underappreciated mechanism of CD28 action. Third, selective CD28 blockade while preserving CTLA-4 creates a regulatory environment through upregulation of Foxp3, PD-1, and IDO, explaining how blockade can promote tolerance rather than merely suppressing activation.

The heterogeneity in experimental systems provides complementary insights. In vitro systems (Jurkat cells, blastoid T cells) 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, and the humanized vasculitis model bridged mechanistic and translational findings.

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 the engagement of alternative costimulatory pathways (CD40-CD154, CD134-CD134L, CD122) that can sustain rejection in CD28’s absence. The efficacy of CD28 blockade thus depends on whether these alternative pathways are simultaneously controlled. Similarly, the differential effects on CD4+ versus CD8+ T cells—with CD40-CD154 and CD134-CD134L blockade being more effective in CD8−/−CD28−/− recipients than CD4−/−CD28−/− recipients—suggests CD4+ T cells have greater dependency on alternative pathways.

The temporal dynamics of pathway alterations also merit attention. Metabolic effects occur within 30 minutes, transcription factor suppression is evident within hours of stimulation, proliferative suppression manifests at 72 hours, and regulatory pathway upregulation persists for at least 100 days. This temporal sequence suggests immediate metabolic and signaling disruptions precede and enable longer-term tolerogenic remodeling.

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, limiting direct species comparisons for these pathways.