CD28 Human
Description
CD28 co-stimulation triggers distinct pathways compared to TCR activation alone :
Core Signaling Events
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PI3K-Akt Pathway: YMNM-dependent activation promotes T cell survival via Bcl-xL upregulation
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NF-κB Activation: Requires coordinated input from both YMNM and proline-rich motifs
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Cytoskeletal Reorganization: Mediated by Nck adaptor protein binding to C-terminal proline residues
Functional Consequences
Species-Specific Signaling Properties
Human CD28 exhibits critical functional differences from murine orthologs:
These differences explain the failed translation of CD28 superagonists from mouse models to human trials .
Therapeutic Targeting Strategies
CD28 modulation represents a frontier in immunotherapy:
Current Approaches
Key Findings from Clinical Studies
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FR104: Achieves >80% CD28 receptor occupancy in primates without cytokine release
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DAb-001: 100-fold greater potency than abatacept against CD86-mediated responses
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CTLA-4-Ig: Reduces IL-2 production by 90% but spares regulatory T cells
Pathological Implications
CD28 dysregulation correlates with:
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Autoimmunity: Reduced CD28<sup>−</sup> T cells in rheumatoid arthritis
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Cancer: CD28 loss in tumor-infiltrating lymphocytes → exhaustion
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Aging: Accumulation of CD28<sup>−</sup> senescent T cells (80% in centenarians)
Emerging Research Directions
Product Specs
T-cell-specific surface glycoprotein CD28, TP44, CD_antigen: CD28, T-cell-specific surface glycoprotein CD28 isoform 1.
Sf9, Baculovirus cells.
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Q&A
What is CD28 and what is its primary function in human T cells?
CD28 is a surface protein expressed on T cells that provides critical costimulatory signals for T cell activation. It interacts with B7.1 (CD80) or B7.2 (CD86) on antigen-presenting cells, and together with signals through the T cell receptor (TCR), provides critical signals for initial T cell activation . At birth, most human T cells express CD28, creating a robust platform for immune response development. Without CD28 costimulation, TCR stimulation typically leads to T cell anergy or apoptosis, highlighting the essential nature of this molecule in proper immune function .
How does CD28 expression change throughout human life?
With age, humans accumulate CD28-negative T cells, particularly in the CD8+ compartment where up to 75% can become CD28-negative in conditions such as HIV infection . Studies across multiple donors show that CD4+ CD28- T cells are found in most individuals (9 of 11 donors in one study) but typically at lower frequencies, ranging from 0% to 46% of the CD3+ CD28- population with a median of 4.4% . The percentage of CD28- T cells remains relatively stable within individuals over time, albeit with some fluctuation, suggesting these represent a distinct population rather than a transient activation state .
What are the phenotypic characteristics of CD28- T cells?
CD28- T cells display several distinctive characteristics:
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They are heterogeneous for CD45RO and CD45RA expression, with a larger proportion expressing CD45RA+ compared to CD28+ T cells
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They have shortened telomeres suggesting extensive proliferative history
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They show a lower threshold for activation, consistent with a memory phenotype
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They produce Th1 cytokines (IFN-γ and TNF-α) but typically not Th2 cytokines like IL-4
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CD28- T cells can be induced to proliferate, acquire effector functions, and increase levels of the survival factor Bcl-XL in response to alternative costimulation, indicating they are not purely senescent
In which pathological conditions are CD28- T cells expanded?
CD28- T cells are notably expanded in several pathological conditions including:
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HIV infection, where up to 75% of the CD8 T cell pool can be CD28-negative
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Rheumatoid arthritis, where loss of CD28 expression on CD4 T cells correlates with disease severity
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Other autoimmune conditions including systemic lupus erythematosus and multiple sclerosis
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Asymptomatic carriers of HCMV, where high frequencies of CD28- T cells with viral specificity are observed
How do CD28- T cells respond to alternative costimulatory pathways?
CD28- T cells can utilize alternative costimulatory pathways, particularly the 4-1BB (CD137)/4-1BBL pathway. Key findings include:
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CD28- T cells rapidly upregulate 4-1BB after TCR stimulation, often faster than CD28+ T cells
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Both CD4+ and CD8+ CD28- subsets express 4-1BB efficiently after stimulation
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4-1BBL costimulation induces proliferation of CD28- T cells, with enhanced responses compared to anti-CD3 alone
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This costimulation pathway increases Bcl-XL protein expression in CD28- T cells, potentially enhancing survival
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4-1BBL stimulation promotes effector cytokine production (IFN-γ and TNF-α) from CD28- T cells
What are the current models explaining the development of CD28- T cells?
Several models explain CD28- T cell development:
Antigen dose model: Studies using EBV peptide/MHC tetramers suggest that T cells specific for latent epitopes (low antigen dose) remain CD45RO+ and CD28+, while those specific for lytic epitopes (high antigen dose) more frequently become CD45RA+ CD28- .
Differentiation state model: Research suggests memory CD8 T cells in chronic viral infections distribute into early, intermediate, and late subsets based on CD28 and CD27 expression, with distribution patterns characteristic of specific viral infections .
Viral-specific distribution: Different chronic viral infections drive distinct patterns of CD28 expression loss, suggesting pathogen-specific mechanisms rather than a universal senescence pathway .
What are the immunotherapeutic implications of targeting CD28?
CD28 targeting presents significant therapeutic opportunities and challenges:
Opportunities:
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CD28 costimulation can enhance T cell responses against tumors when combined with CD3 targeting approaches
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New non-superagonistic anti-CD28 antibodies provide safer alternatives to previous approaches
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Combinatorial therapy with CD3 bispecific antibodies shows enhanced tumor cell killing and T-cell proliferation
Challenges:
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Historical safety concerns after the TeGenero Phase 1 trial with TGN1412 resulted in severe cytokine release syndrome
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Careful epitope selection is critical - superagonistic antibodies binding to lateral epitopes versus conventional antibodies binding apical epitopes like natural ligands
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The heterogeneity of CD28 expression in aging or diseased individuals may affect therapeutic efficacy
What methods are most effective for studying CD28- T cell populations?
For accurate investigation of CD28- T cells:
Isolation approaches:
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Fluorescence-activated cell sorting (FACS) using anti-CD3 and anti-CD28 antibodies achieves >96% purity
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When isolating CD28- T cells, contamination with even small numbers of CD28+ T cells can significantly affect results
Characterization protocols:
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4-color flow cytometry allows simultaneous analysis of CD28, CD3, CD4/CD8, and functional markers
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Analysis of memory/naive markers (CD45RA/RO) helps stratify CD28- subpopulations
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Functional assays should include proliferation (CFSE dilution), cytokine production (intracellular cytokine staining), and survival factor expression
How should researchers design experiments to study costimulatory requirements of CD28- T cells?
Experimental design considerations:
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Use plate-bound anti-CD3 at standardized concentrations for TCR stimulation
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For costimulatory studies, employ transfected cell lines expressing costimulatory ligands (e.g., P815 cells expressing 4-1BBL)
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Control P815 cells and P815-4-1BBL-transfected cell lines should bind equivalent levels of anti-CD3 to ensure comparable primary stimulation
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When analyzing separated populations, verify purity (typically >95%)
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Consider that optimal effects of costimulatory ligands like 4-1BBL may require CD28+ T cells to provide IL-2
What approaches are recommended for developing safe CD28-targeting therapeutics?
Design strategies:
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Use phage display technology with strategic antigen presentation to favor isolation of binders toward specific domains
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Target binding epitopes near the apex of CD28 (similar to natural ligands) rather than lateral epitopes (as in superagonistic antibodies like TGN1412)
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Biotinylate recombinant CD28 at the membrane-proximal part to favor the isolation of binders toward the apex
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Verify homogeneity of recombinant proteins by size exclusion chromatography (SEC) and SDS-PAGE
Safety assessment:
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Screen for in vitro superagonistic properties using human PBMCs from multiple donors
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Conduct in vivo safety studies in humanized NSG mice to assess potential cytokine release syndrome
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Compare directly with known superagonistic antibodies (like TGN1412) as controls
What analytical techniques provide the most insight into CD28 function?
Recommended analytical approaches:
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Epitope mapping to determine the conformational binding epitope of anti-CD28 antibodies
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Flow cytometry on primary human and mouse T-cells to confirm binding specificity
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Assessment of T-cell activation markers without TCR/CD3 stimulation to identify potential superagonistic properties
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Measurement of in vitro activity using human PBMCs in combination with CD3 bispecific antibodies to evaluate enhancement of tumor cell killing and T-cell proliferation
How should researchers interpret variability in CD28 expression across donors?
When analyzing CD28 expression variability:
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No significant correlation exists between the percentage of CD28- T cells and donor age or sex across a limited age range (23-55 years)
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In most donors, the CD8+ CD28- population predominates, often comprising >95% of CD28- T cells
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CD4+ CD28- T cell frequencies vary widely between individuals (0-46% of CD3+ CD28- cells)
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The percentage of CD28- T cells generally remains stable within donors over time, though with some fluctuation
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When designing studies, test across multiple donors with varying CD28- T cell frequencies to account for this heterogeneity
What are key considerations when interpreting functional studies of CD28- T cells?
Interpretation guidelines:
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CD28- T cells show a small amount of cell division in response to anti-CD3 alone, while CD28+ T cells are unresponsive to TCR signaling without costimulation
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This suggests CD28- T cells have a lower activation threshold, consistent with a memory phenotype
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When analyzing cytokine production, note that CD28- T cells produce lower levels of TNF-α than CD28+ T cells
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CD28- T cells fail to produce the Th2 cytokine IL-4, showing a Th1-biased functional profile
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The CD28- memory T cell population is unlikely to represent a purely senescent population of T cells given their functional capabilities
What are promising approaches for targeting CD28 in next-generation immunotherapies?
The development of novel CD28-targeting agents shows significant promise:
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Fully human non-superagonistic anti-CD28 antibodies like E1P2 represent a new class of therapeutics with improved safety profiles
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Epitope selection is crucial: binding near the apex of CD28 (similar to natural ligands) rather than lateral epitopes may prevent superagonistic effects
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Combination therapy with CD3 bispecific T-cell engagers may provide optimal T-cell activation while preventing early exhaustion
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These approaches show particular promise for enhancing immunotherapeutic approaches against cancer or infectious diseases
How might understanding CD28- T cells impact personalized medicine approaches?
The heterogeneity of CD28 expression across individuals suggests important considerations for personalized medicine:
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Patient stratification based on CD28 expression patterns may predict response to immunotherapies
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In conditions with expanded CD28- T cell populations (HIV, autoimmunity), alternative costimulatory targeting may be necessary
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Age-related accumulation of CD28- T cells may impact immunotherapy efficacy in older patients
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The development of combinatorial approaches targeting both CD3 and costimulatory receptors may be essential for optimal therapeutic responses in patients with significant CD28- T cell populations
Product Science Overview
CD28 functions as a co-stimulatory receptor that is essential for the full activation of T cells. It works in conjunction with the T-cell receptor (TCR) to amplify TCR signals and deliver unique signals that control intracellular biochemical events, ultimately altering the gene expression program of T cells . This co-stimulatory pathway is vital for the regulation of T and B cell responses, and it involves interactions with its ligands, B7-1 (CD80) and B7-2 (CD86) .
Recombinant human CD28 is produced using advanced biotechnological methods. The DNA sequence encoding human CD28 is expressed in host cells, such as HEK293 cells, to produce the recombinant protein. The recombinant human CD28 protein is often biotinylated and verified through various methods, including HPLC and MALS . The recombinant protein is typically provided as a lyophilized powder and can be reconstituted for use in various research applications .
Recombinant human CD28 is widely used in immunological research to study T-cell activation and co-stimulation. It is also utilized in the development of immunotherapies and in the investigation of immune responses. The ability to produce recombinant CD28 allows researchers to explore its functions and interactions in a controlled environment, providing valuable insights into immune regulation and potential therapeutic targets.
