CD80 Mouse

What is the expression pattern of CD80 in mouse immune cells?

In mice, CD80 is primarily expressed on antigen-presenting cells (APCs) including monocytes, peritoneal macrophages, and dendritic cells. Expression can be significantly increased on B lymphocytes following stimulation with lipopolysaccharide (LPS) and interleukin-4 (IL-4) . Importantly, CD80 expression patterns differ between cell populations and activation states. While naïve CD8+ T cells do not display CD80, effector and memory CD8+ T cells show significant CD80 surface expression following acute lymphocytic choriomeningitis virus (LCMV) infection .

The expression pattern can be categorized as follows:

• Constitutive expression: Detected on various antigen-presenting cells

• Inducible expression: Upregulated on B cells following specific stimuli

• Acquired expression: Observed on effector and memory CD8+ T cells through both intrinsic expression and extrinsic acquisition mechanisms

How can CD80 be reliably detected in mouse models?

Multiple complementary techniques can be employed for robust CD80 detection:

Flow cytometry: The gold standard for surface expression analysis. CD80 can be detected on mouse splenocytes using specific antibodies such as Goat Anti-Mouse B7-1/CD80 followed by appropriate secondary antibodies. Signal enhancement is possible by stimulating splenocytes with 200 ng/mL LPS for 48 hours prior to staining . This approach allows for multiparametric analysis, enabling simultaneous assessment of CD80 with other markers like B220/CD45R .

Western blot: For total protein detection, lysates from cells like C2C12 mouse myoblast can be probed with anti-CD80 antibodies. CD80 typically appears as a band at approximately 60 kDa under reducing conditions . This method quantifies total protein rather than surface expression.

Immunohistochemistry: For tissue localization studies, particularly useful for examining CD80 distribution in lymphoid organs versus peripheral tissues .

When comparing different tissues, note that CD80 extrinsic acquisition by CD8+ T cells is observed primarily in lymphoid organs but not in peripheral tissues, necessitating appropriate tissue sampling strategies .

What are the mechanisms of extrinsic acquisition of CD80 by T cells in vivo?

The extrinsic acquisition of CD80 by T cells occurs through a process called trogocytosis, which is a cell-contact dependent uptake of plasma membrane components and associated molecules . This process has distinct characteristics in different T cell populations:

Effector CD8+ T cells: Display CD80 through both intrinsic expression and extrinsic acquisition mechanisms. During acute LCMV infection, effector CD8+ T cells upregulate CD80 expression during the early differentiation phase .

Memory CD8+ T cells: Display CD80 exclusively through extrinsic acquisition, not intrinsic expression. This acquisition is anatomically restricted to lymphoid organs and not observed in peripheral tissues .

To experimentally investigate this process, researchers can employ adoptive transfer models using CD80-knockout (KO) CD8+ T cells transferred into wild-type recipients. This approach allows for clear discrimination between intrinsic expression and extrinsic acquisition by tracking CD80 appearance on CD80-KO donor cells, which can only acquire CD80 extrinsically from host cells .

The anatomical restriction of CD80 acquisition suggests specialized microenvironmental requirements for trogocytosis, likely involving specific cell-cell interactions within lymphoid structures .

How does CD80 on memory CD8+ T cells influence recall immune responses?

CD80 molecules displayed on memory CD8+ T cells play a regulatory role in recall immune responses by:

• Limiting expansion: Memory CD8+ T cells that have extrinsically acquired CD80 demonstrate reduced proliferative responses upon secondary antigen challenge compared to CD80-deficient memory CD8+ T cells .

• Reducing IL-2 production: The presence of CD80 on memory CD8+ T cells is associated with decreased IL-2 production during recall responses .

This inhibitory effect can be experimentally demonstrated by comparing recall responses between:

• Memory CD8+ T cells that have extrinsically acquired CD80 (from wild-type recipients)

• CD80-deficient memory CD8+ T cells (from CD80-KO recipients)

The methodological approach involves:

• Adoptive transfer of CD80-KO P14 CD8+ T cells into either wild-type or CD80-KO recipients

• Primary infection with LCMV to generate memory CD8+ T cells

• Isolation of memory cells after day 60 post-infection

• Secondary transfer of these memory cells into new recipients

• Challenge with LCMV and assessment of expansion and cytokine production

This negative regulatory function suggests CD80 on memory CD8+ T cells may help prevent excessive immune responses during secondary challenges.

What are the optimal experimental approaches to study CD80's role in T cell function?

Rigorous investigation of CD80's role in T cell function requires sophisticated experimental designs:

Adoptive transfer models with congenic markers: Use of wild-type and CD80-KO T cells with different congenic markers (e.g., Thy1.1/1.1 and Thy1.1/1.2) allows for precise tracking of cell populations and discrimination between intrinsic expression and extrinsic acquisition . This approach enables:

• Simultaneous comparison of different cell populations within the same host

• Minimization of inter-animal variability

• Precise quantification of cell expansion and function

Timing considerations: The dynamics of CD80 expression change dramatically over the course of an immune response. Key timepoints include:

• Effector phase (days 8-15 post-infection): Both intrinsic expression and extrinsic acquisition occur

• Memory phase (day 60+ post-infection): Primarily extrinsic acquisition

Tissue sampling strategy: Given that CD80 extrinsic acquisition occurs in lymphoid organs but not peripheral tissues, comprehensive analysis should include:

• Spleen and lymph nodes (primary sites of acquisition)

• Peripheral tissues (negative control for acquisition)

• Blood (for monitoring dynamic changes)

Functional readouts: Beyond surface expression, assess:

• Proliferation (using CFSE dilution or congenic marker frequency)

• Cytokine production (particularly IL-2)

• Memory recall responses (upon secondary challenge)

How can researchers effectively isolate and characterize CD80-expressing versus CD80-deficient memory CD8+ T cells?

Isolation and characterization of CD80-expressing versus CD80-deficient memory CD8+ T cells requires a sequential approach:

• Generation of memory populations:

  • Adoptive transfer of CD80-KO P14 CD8+ T cells into either wild-type (for CD80-acquired) or CD80-KO (for CD80-deficient) recipients

  • Infection with 2×10^5 PFUs of LCMV Arm intraperitoneally

  • Allow 60+ days for memory formation

• Isolation procedure:

  • Harvest spleens from mice at day 60+ post-infection

  • Prepare single-cell suspensions

  • Isolate total CD8+ T cells using negative selection (MACS technology)

  • Further purify memory P14 cells using fluorescence-activated cell sorting based on congenic markers (e.g., Thy1.1)

• Quality control criteria:

  • Purity: >95% specific memory cells

  • Viability: >90% viable cells

  • Phenotype: CD44^high CD62L^high/low (central/effector memory)

  • Functionality: Capable of rapid IFN-γ production upon stimulation

• Characterization panel:

  • Surface markers: CD80, CD44, CD62L, CD127, KLRG1

  • Functional assays: Cytokine production, proliferative capacity

  • Secondary challenge response analysis

This approach allows for direct comparison of memory CD8+ T cell functions with and without acquired CD80, providing insights into CD80's regulatory role in recall responses.

How does CD80 influence viral infection outcomes in mouse models?

CD80 plays complex roles in viral infection outcomes through multiple mechanisms:

In HSV-1 infection models:

• Overexpression of CD80 leads to productive infection in normally non-permissive dendritic cells in vitro

• HSV-1 recombinant virus expressing CD80 (HSV-CD80) causes more severe eye disease

• CD80 expression is detected on the surface of infected cells

• Transcriptome analysis reveals similar viral gene expression patterns between HSV-CD80 and parental virus, despite differences in disease severity

In LCMV infection models:

• CD80 expression is upregulated on antigen-specific CD8+ T cells following both acute and chronic LCMV infection

• The percentage of CD80-expressing cells is higher in CD44^high CD8+ T cells compared to CD44^high CD4+ T cells

• CD80 upregulation is maintained on memory or exhausted CD8+ T cells long-term, regardless of LCMV strain

The dual role of CD80 in promoting protective immunity while potentially exacerbating immunopathology represents a critical balance point in infection outcomes. Researchers should consider both direct viral control mechanisms and immunopathological consequences when assessing CD80's role in viral infections.

What are the functional implications of CD80-CD28 versus CD80-CTLA-4 interactions in mouse immunological studies?

CD80 engages in two critical but opposing interactions with significant functional implications:

CD80-CD28 interaction:

• Provides co-stimulatory signals for T cell activation

• Stimulates and sustains T cell responses

• Promotes IL-2 secretion in a dose-dependent manner

• Can be experimentally demonstrated using recombinant Mouse B7-1/CD80 Fc Chimera, which co-stimulates IL-2 secretion in human T cell lines in the presence of PHA

CD80-CTLA-4 interaction:

• Provides inhibitory signals

• Suppresses T cell responses

• Contributes to peripheral tolerance

• Has higher binding affinity than CD80-CD28 interaction

These opposing functions can be experimentally investigated using:

• Blocking antibodies: Anti-CD80 antibodies can neutralize IL-2 secretion elicited by recombinant mouse B7-1/CD80 Fc Chimera, with ND50 typically 0.15-0.6 μg/mL

• Genetic approaches: Using CD80-KO mice or cells to examine the consequences of CD80 absence

• Targeted blockade: Selectively blocking either CD28 or CTLA-4 to dissect the relative contributions of each pathway

Understanding the balance between these interactions is crucial for interpreting experimental results and developing targeted immunomodulatory approaches.

How should researchers address discrepancies in CD80 expression data between different experimental systems?

Discrepancies in CD80 expression data between different experimental systems are common and require systematic analysis:

Sources of variability to consider:

• Strain differences: Different mouse strains may exhibit baseline variations in CD80 expression and regulation

• Tissue-specific effects: CD80 expression and acquisition patterns differ markedly between lymphoid organs and peripheral tissues

• Temporal dynamics: CD80 expression changes significantly over time post-infection or stimulation

• Technical factors: Antibody clones, fluorochromes, and detection methods can influence results

• Activation state: CD80 expression is highly dependent on cellular activation state

Recommended approach for reconciling discrepancies:

• Standardize experimental conditions: Use consistent stimulation protocols, timing, and detection methods

• Use multiple detection methods: Combine flow cytometry with Western blot and/or qPCR

• Include appropriate controls: Both positive controls (LPS-stimulated splenocytes) and negative controls (CD80-KO cells)

• Perform titration experiments: Establish dose-response relationships for stimuli

• Consider microenvironmental factors: Account for interactions with other cell types that may influence CD80 expression

• Statistical analysis: Use appropriate statistical tests that account for biological variability

When analyzing CD80 expression on T cells specifically, always distinguish between intrinsic expression and extrinsic acquisition by using appropriate experimental designs with CD80-KO donor cells in wild-type recipients .

What statistical approaches are most appropriate for analyzing CD80 acquisition and function in complex immunological experiments?

Complex immunological experiments investigating CD80 acquisition and function require sophisticated statistical approaches:

For analyzing CD80 acquisition kinetics:

• Repeated measures ANOVA: Appropriate for time-course experiments tracking CD80 expression on the same cell populations over multiple time points

• Mixed-effects models: Useful when combining data from multiple experiments with potential batch effects

• Area under curve (AUC) analysis: For comparing cumulative CD80 expression profiles between experimental groups

For functional comparisons:

• Two-tailed unpaired Student's t-test: Commonly used for comparing two independent groups, such as CD80-acquired versus CD80-deficient memory T cells

• ANOVA with post-hoc tests: For experiments with multiple comparison groups

• Non-parametric alternatives: When data do not meet normality assumptions (Mann-Whitney, Kruskal-Wallis)

Considerations for experimental design and analysis:

• Power calculations: Determine appropriate sample sizes based on expected effect sizes from preliminary data

• Paired analyses: Use paired statistical tests when comparing donor populations within the same recipient

• Multiple testing correction: Apply Bonferroni or false discovery rate corrections when performing multiple comparisons

• Visualization approaches: Present data using appropriate graphical formats (e.g., GraphPad Prism software)

• Biological versus technical replicates: Clearly distinguish between these in experimental design and analysis