CD274 Mouse

What is CD274 and what are its alternative designations in mouse research?

CD274, commonly known as Programmed Death Ligand 1 (PD-L1), is a 40 kD type I transmembrane protein belonging to the B7 family within the immunoglobulin receptor superfamily. In mouse research literature, CD274 is also referred to by several alternative names including B7-H1, PDCD1LG1, PDCD1L1, and PDL1. This molecule is encoded by the Cd274 gene located on chromosome 9p24 in humans, with the mouse ortholog having the Entrez Gene ID 60533. The protein functions as a ligand for PD-1 (CD279) and plays critical roles in T cell regulation, particularly in inhibiting T cell responses under certain conditions, though it can also provide costimulatory signals in some contexts .

How does CD274 expression vary across different mouse immune cell populations?

CD274 expression in mice demonstrates significant cell type specificity and activation-dependent patterns:

• T cells and B cells: Constitutive low-level expression with upregulation upon activation

• Natural Killer (NK) cells: Expression observed particularly after activation

• Dendritic cells: Both immature and mature dendritic cells express CD274, with higher levels in mature DCs

• Monocytes/Macrophages: Expression increases significantly upon IFN-γ stimulation

• Non-immune tissues: Expression can be induced in endothelial cells and various tissue cells during inflammation

The expression of CD274 is dynamically regulated in response to inflammatory signals, with IFN-γ being a particularly potent inducer across multiple cell types. Flow cytometric analysis of mouse peripheral blood mononuclear cells often shows heterogeneous expression patterns depending on the activation state of the cells .

What are the recommended methods for detecting CD274 in mouse samples?

Several validated methods exist for detecting CD274 in mouse samples:

• Flow Cytometry: The most common approach uses fluorochrome-conjugated antibodies (e.g., PE, APC, Brilliant Violet 421™) against mouse CD274. Clone 10F.9G2 is widely used for mouse CD274 detection with high specificity .

• Immunohistochemistry/Immunofluorescence: For tissue sections or fixed cells, purified anti-CD274 antibodies followed by appropriate secondary detection systems are recommended.

• Western Blotting: For protein expression analysis, though optimization of lysis conditions is crucial for this transmembrane protein.

• RT-PCR/qPCR: For mRNA expression analysis when protein detection is challenging or to complement protein studies.

Best practices include:

• Using appropriate isotype controls

• Including BD Horizon Brilliant™ Stain Buffer when using BD Horizon Brilliant dyes to minimize fluorescent dye interactions

• Creating compensation controls with either cells or beads when performing multicolor flow cytometry .

What are the primary immunological functions of CD274 in mice?

CD274 in mice serves several critical immunological functions:

• T cell response regulation: CD274 interaction with PD-1 inhibits T cell proliferation and cytokine production, serving as a checkpoint to prevent excessive immune activation .

• Dual signaling capability: Interestingly, CD274 can also costimulate T cell growth and cytokine production in an IL-2-dependent manner that is PD-1-independent .

• Homeostatic immune regulation: During infection or inflammation in normal tissues, CD274-PD-1 interaction prevents autoimmunity by maintaining immune homeostasis .

• Thymocyte development: CD274 plays a role during development by inhibiting TCR-mediated positive selection of thymocytes .

• CD4+ T cell regulation: It has specific functions in regulating CD4 positive T cell proliferation .

These functions highlight CD274's complex role as both an inhibitory and potentially stimulatory molecule depending on the context, differentiation state of the responding T cells, and the presence of other signaling pathways .

How does mouse CD274 compare to human CD274 in structure and function?

While mouse and human CD274 share significant homology, several key differences exist:

These similarities and differences should be considered when translating findings from mouse models to human applications. While the core functions of inhibiting T cell responses and providing immune regulation are conserved, species-specific differences may influence therapeutic targeting strategies and interpretation of experimental results in tumor microenvironments .

What are the validated approaches for CD274 knockout in mouse models?

Several validated approaches exist for generating CD274 knockout mice:

• CRISPR/Cas9 gene editing: Currently the most efficient method, utilizing guide RNAs targeting the Cd274 gene (ID: 60533). Commercial CRISPR knockout kits typically include:

  • Multiple gRNA vectors targeting different regions of the gene

  • A linear donor with a selection cassette (e.g., LoxP-EF1A-tGFP-P2A-Puro-LoxP)

  • Validation primers and protocols

• Conventional homologous recombination: Traditional approach using targeting vectors with homology arms flanking a selection cassette.

• Conditional knockout strategies: Using Cre-loxP or similar systems to achieve tissue-specific or inducible CD274 deletion.

Important considerations when generating CD274 knockout mice include:

• Confirming complete deletion at both genomic and protein levels

• Assessing potential compensatory upregulation of PD-L2 (CD273)

• Monitoring for spontaneous autoimmune manifestations that may affect experimental interpretations

What antibody clones are recommended for blocking mouse CD274 function in vivo?

Several validated antibody clones are available for blocking mouse CD274 function in vivo:

*Note: 29E.2A3 is specific for human PD-L1 and included for reference

For in vivo blocking experiments:

• Ultra-LEAF™ purified antibody preparations are recommended to minimize endotoxin contamination

• Typical dosing ranges from 100-200 μg per mouse every 3-4 days

• Control groups should receive matched isotype control antibodies

• Validation of PD-L1 blockade should be performed via flow cytometry or functional assays

What technical considerations are important for flow cytometric analysis of CD274 in mouse samples?

When analyzing CD274 expression by flow cytometry in mouse samples, several technical considerations are crucial:

• Sample preparation protocols:

  • Fresh samples yield optimal results, though proper fixation can preserve CD274 epitopes

  • Enzymatic tissue dissociation may affect certain epitopes; mechanical dissociation is preferable when possible

  • Red blood cell lysis should use gentle methods to preserve CD274 expression

• Antibody panel design:

  • CD274 expression should be assessed in the context of lineage markers to identify specific expressing populations

  • When using BD Horizon Brilliant™ fluorochromes, include BD Horizon Brilliant Stain Buffer to minimize fluorescent dye interactions

  • Consider potential spectral overlap, particularly when using PE, PE/Cy7 or BV421 conjugates

• Controls and standardization:

  • Include FMO (Fluorescence Minus One) controls for accurate gating

  • Use compensation controls with either cells or beads exposed to the same staining buffer

  • Consider using standardized beads to normalize between experiments

• Analysis considerations:

  • CD274 expression is often heterogeneous; consider population frequency and mean fluorescence intensity

  • Activation status dramatically affects expression; standardize activation conditions

  • Human donor-specific background related to anti-PEG antibodies may occur due to COVID-19 vaccines or other PEG-containing products

What phenotypes are observed in CD274-deficient mice in various disease models?

CD274-deficient mice exhibit several notable phenotypes across different disease models:

• Autoimmune manifestations:

  • Increased susceptibility to experimental autoimmune encephalomyelitis (EAE)

  • More severe collagen-induced arthritis

  • Enhanced autoantibody production in some genetic backgrounds

• Infection responses:

  • Improved clearance of certain chronic viral infections

  • Potentially fatal immunopathology during acute infections due to excessive T cell responses

  • Enhanced T cell memory formation following pathogen exposure

• Tumor immunity:

  • Reduced tumor growth rates in multiple cancer models

  • Increased tumor-infiltrating lymphocyte activity

  • Enhanced efficacy of cancer vaccines

• Reproduction and development:

  • Decreased allogeneic fetal survival rates, suggesting a role in maternal-fetal tolerance

  • Normal development in the absence of other immune challenges

These phenotypes highlight CD274's critical role in maintaining immune homeostasis, particularly in the context of chronic antigen exposure or inflammatory conditions .

How does manipulation of CD274 expression impact anti-tumor immunity in mouse cancer models?

Manipulation of CD274 expression has profound effects on anti-tumor immunity in mouse cancer models:

• CD274 blockade effects:

  • Enhanced CD8+ T cell infiltration into tumors

  • Increased production of effector cytokines (IFN-γ, TNF-α)

  • Reduced T cell exhaustion phenotypes within the tumor microenvironment

  • Synergistic effects when combined with other checkpoint inhibitors (e.g., CTLA-4 blockade)

• Genetic manipulation findings:

  • Tumors engineered to overexpress CD274 show accelerated growth and immune evasion

  • CD274 knockout in tumor cells renders them more susceptible to immune-mediated rejection

  • miR-155 in T cells appears necessary to limit melanoma growth, potentially by regulating PD-1/PD-L1 axis

• Combination approaches:

  • CD274 blockade combined with radiotherapy shows enhanced efficacy due to increased tumor antigen presentation

  • Targeting CD274 along with immunostimulatory agents (e.g., TLR agonists) produces synergistic anti-tumor effects

  • Sequential blocking of different checkpoints may overcome resistance mechanisms

These findings have directly informed human clinical trial designs and highlight the importance of CD274 as a key immune evasion mechanism employed by tumors .

What methodological approaches are recommended for studying CD274's role in the tumor microenvironment?

To effectively study CD274's role in the tumor microenvironment, several methodological approaches are recommended:

• Spatial analysis techniques:

  • Multiplex immunofluorescence to simultaneously visualize CD274 with immune cell markers

  • Laser capture microdissection of CD274+ regions followed by transcriptomic analysis

  • In situ hybridization combined with protein detection to correlate mRNA and protein expression

• Functional assessment protocols:

  • Ex vivo tumor slice cultures to preserve spatial relationships while testing CD274 blockade

  • Adoptive transfer of CD274-deficient vs. wild-type immune cells into tumor-bearing mice

  • Dual reporter systems to simultaneously track T cell activation and CD274 expression dynamics

• Single-cell analysis approaches:

  • Single-cell RNA sequencing of tumor-infiltrating immune cells with computational deconvolution of PD-1/PD-L1 interactions

  • CyTOF (mass cytometry) panels incorporating CD274 and multiple immune checkpoint molecules

  • Live-cell imaging of T cell/tumor cell interactions in the presence/absence of CD274 blockade

• Experimental design considerations:

  • Include multiple tumor models with varying baseline CD274 expression

  • Assess temporal dynamics of CD274 expression during tumor progression

  • Consider combination approaches targeting multiple immune checkpoints or immunosuppressive mechanisms

These methodological approaches provide complementary information about CD274's spatial distribution, regulatory mechanisms, and functional impact within the complex tumor microenvironment .

What factors can influence CD274 detection in mouse samples and how can they be addressed?

Several factors can significantly influence CD274 detection in mouse samples:

• Sample preparation variables:

  • Cell isolation method: Enzymatic digestion can cleave epitopes; optimize digestion time or use mechanical dissociation

  • Freezing/thawing cycles: Limit to preserve membrane protein integrity

  • Fixation: Paraformaldehyde concentrations above 2% may reduce epitope accessibility

  Solution: Standardize preparation protocols and include positive controls from known CD274-expressing cell populations

• Biological variables:

  • Activation status: CD274 expression increases dramatically after activation

  • Cytokine environment: IFN-γ particularly upregulates CD274

  • Time since stimulation: Expression peaks at different timepoints depending on cell type

  Solution: Document timing and activation conditions precisely; consider time-course experiments

• Technical variables:

  • Antibody clone compatibility with applications

  • Fluorochrome brightness and spectral overlap

  • Buffer composition affecting antibody binding

  Solution: Use BD Horizon Brilliant™ Stain Buffer with brilliant dyes; titrate antibodies; use appropriate isotype controls

How can researchers distinguish between CD274 (PD-L1) and CD273 (PD-L2) in mouse studies?

Distinguishing between CD274 (PD-L1) and CD273 (PD-L2) in mouse studies requires careful consideration of several approaches:

• Antibody selection strategy:

  • Use monoclonal antibodies with validated specificity (e.g., clone 10F.9G2 for CD274)

  • Perform validation using cells from knockout mice for each molecule

  • Consider including both markers in the same panel with spectrally distinct fluorochromes

• Expression pattern analysis:

  • CD274 is broadly expressed on multiple immune and non-immune cells

  • CD273 has a more restricted expression pattern, primarily on dendritic cells and macrophages

  • Differential regulation: CD273 and CD274 respond differently to various stimuli

• Functional assessment approaches:

  • Use specific blocking antibodies to each ligand

  • Employ genetic knockouts of each ligand individually

  • Conduct competitive binding assays with recombinant proteins

• Molecular techniques:

  • Design PCR primers specific to unique regions of each gene

  • Use specific siRNA/shRNA constructs targeting each molecule

  • Employ CRISPR/Cas9 systems with guides specifically designed for each target

These approaches ensure accurate attribution of biological effects to the correct PD-1 ligand in experimental systems .

What emerging technologies are advancing CD274 research in mouse models?

Several cutting-edge technologies are revolutionizing CD274 research in mouse models:

• Advanced genetic engineering approaches:

  • Base editing for introducing specific point mutations in CD274

  • Prime editing for precise modifications without double-strand breaks

  • Inducible CRISPR systems for temporal control of CD274 deletion

  • Tissue-specific promoters driving CD274 expression in targeted cell populations

• Innovative imaging technologies:

  • Intravital microscopy to visualize PD-1/PD-L1 interactions in living tissues

  • Optical clearing techniques combined with light-sheet microscopy for whole-organ CD274 mapping

  • CODEX multiplexed imaging for simultaneous detection of dozens of markers alongside CD274

  • PET imaging with radiolabeled anti-CD274 antibodies for whole-body distribution studies

• Single-cell and spatial omics:

  • Spatial transcriptomics to map CD274 expression in tissue context

  • Single-cell ATAC-seq to identify regulatory elements controlling CD274 expression

  • Multi-modal single-cell approaches combining protein, RNA and chromatin accessibility

  • Spatial proteomics to identify CD274 interaction partners in different cellular compartments

• Computational biology approaches:

  • Machine learning algorithms to predict CD274 expression based on other cellular parameters

  • Systems biology modeling of the PD-1/PD-L1 signaling network

  • Virtual screening for novel small molecule modulators of CD274

These technologies promise to provide unprecedented insights into CD274 biology and therapeutic targeting .

What are the emerging strategies for therapeutic targeting of CD274 being tested in mouse models?

Innovative strategies for therapeutic targeting of CD274 under investigation in mouse models include:

• Bispecific antibody approaches:

  • PD-L1 x CD3 bispecifics to redirect T cells to PD-L1+ cells

  • PD-L1 x CD47 bispecifics to simultaneously block "don't eat me" signals

  • PD-L1 x costimulatory receptor (e.g., 4-1BB) bispecifics to convert inhibitory signals to stimulatory ones

• Small molecule inhibitors:

  • Compounds targeting the PD-1/PD-L1 interaction interface

  • Molecules affecting CD274 protein stability or trafficking

  • Inhibitors of signaling pathways regulating CD274 expression

• Nucleic acid-based therapies:

  • siRNA and antisense oligonucleotides targeting CD274 mRNA

  • mRNA vaccines encoding modified CD274 to generate anti-PD-L1 immune responses

  • CRISPR-based approaches to modulate CD274 expression in specific cell populations

• Combination immunotherapy strategies:

  • CD274 blockade with agonists of costimulatory receptors (ICOS, OX40, 4-1BB)

  • Sequential or alternating checkpoint blockade regimens

  • Integration with conventional therapies (radiation, chemotherapy) for enhanced immunogenic cell death

These approaches are being rigorously tested in mouse models before potential translation to human clinical trials, with careful assessment of efficacy, toxicity profiles, and mechanisms of action .