BTLA Mouse

What is BTLA and what is its role in mouse immune system?

BTLA is a co-inhibitory receptor that negatively regulates B and T cell activation in mice. It plays a crucial role in maintaining immune tolerance and preventing excessive immune responses. BTLA is expressed on various immune cells, including CD4+ T cells, CD8+ T cells, and B cells . Research shows that BTLA deficiency exacerbates lupus disease progression in mouse models , highlighting its importance in preventing autoimmunity.

BTLA functions by providing inhibitory signals that limit lymphocyte activation, proliferation, and effector functions. The receptor contains immunoreceptor tyrosine-based inhibitory motifs (ITIMs) in its cytoplasmic domain, which recruit phosphatases to dampen immune cell signaling .

How is BTLA expression distributed across mouse immune cell populations?

BTLA expression varies across different immune cell subsets in mice:

This distribution is important when designing experiments targeting specific cell populations for BTLA-related studies.

What techniques are commonly used to detect BTLA in mouse tissues?

Several methodological approaches are available for detecting BTLA in mouse tissues:

• Western Blot Analysis: As described in search result , Western blot can detect BTLA in mouse spleen and lymph node tissues. PVDF membrane probed with 1 μg/mL of Goat Anti-Mouse BTLA Antigen Affinity-purified Polyclonal Antibody (Catalog # AF3007) followed by HRP-conjugated secondary antibody reveals a specific band at approximately 60 kDa .

• Flow Cytometry: For quantitative assessment of BTLA expression on cell surfaces of different immune populations.

• Single-Cell Analysis: Mentioned in search result , this technique provides detailed expression patterns at the individual cell level.

• Reverse Phase Protein Array (RPPA): Used to evaluate downstream signaling of BTLA in mouse T cells .

Experimental protocol for Western blot detection typically includes:

• Tissue lysate preparation from mouse spleen or lymph nodes

• SDS-PAGE under reducing conditions

• Transfer to PVDF membrane

• Probing with anti-BTLA antibody (1 μg/mL)

• Detection with appropriate secondary antibody

• Visualization using Immunoblot Buffer Group 1

How does BTLA signaling affect mouse T cell activation?

BTLA provides inhibitory signals that regulate T cell activation in mice. When engaged, BTLA inhibits:

• Upregulation of activation markers (CD69, CD25)

• Proliferation of T cells

• Cytokine production

Functional studies show that BTLA's inhibitory capacity is impaired in diseased NZB/W lupus mice compared to control BALB/W mice, particularly in CD4+ T cells . This dysfunction manifests as decreased ability to inhibit CD69/CD25 expression and reduced anti-proliferative effects .

The inhibitory function of BTLA requires proper recruitment to TCR clusters. In diseased NZB/W mice, TCR pre-clusters can be visualized even without stimulation, but BTLA is often not properly recruited to these structures, leading to defective inhibitory signaling .

What phenotypic changes occur in BTLA knockout mice?

BTLA knockout mice exhibit several important immunological changes:

• Enhanced T cell responses to antigenic stimulation

• Increased susceptibility to autoimmune diseases

• Exacerbated lupus disease progression

• Altered immune cell homeostasis

These phenotypic changes highlight BTLA's critical role in maintaining immune balance and preventing autoimmunity.

How does BTLA function differ between naive and memory CD4+ T cells in disease models?

A significant functional difference exists in BTLA-mediated inhibition between naive and memory CD4+ T cells in lupus-prone NZB/W mice:

Notably, when naive CD4+ T cells from old-diseased NZB/W mice were isolated and tested separately, they maintained normal BTLA functionality . This suggests that the impaired BTLA function observed in total CD4+ T cells is primarily associated with the expanded effector/memory compartment rather than representing a global defect .

What is the mechanistic explanation for impaired BTLA functionality in autoimmune mouse models?

The impaired BTLA functionality in autoimmune mouse models involves several mechanisms:

How does therapeutic targeting of BTLA affect disease progression in lupus-prone mice?

Targeting BTLA with the anti-BTLA antibody 6F7 shows significant therapeutic potential in lupus-prone mice:

Regarding the 6F7 antibody's mechanism of action:

• It is not a depleting antibody

• Does not block HVEM binding to BTLA

• Induces BTLA down-modulation

• Exhibits in vivo agonist activity

Importantly, relapse occurred rapidly after the loss of BTLA receptor occupancy, suggesting the antibody's beneficial effect requires ongoing stimulation of the BTLA pathway rather than inducing permanent tolerance .

What techniques are available for manipulating BTLA expression in experimental mouse models?

Multiple methodological approaches exist for manipulating BTLA expression in mouse models:

• Retroviral Transduction of BTLA-KO T Cells:

  • Protocol outlined in search result :
    a. Co-transfection of pRVKM retroviral vectors and pEco plasmids into Plate-E cells
    b. Harvesting supernatants after 60h and concentration using Vivaspin-20
    c. Activation of splenocytes from BTLA-KO mice with anti-mouse CD3 (0.3 μg/ml) for 24h
    d. Infection with concentrated retrovirus
    e. Expansion in media with hIL-2 for 3 days
    f. Cell sorting based on GFP expression
    g. Further propagation with hIL-2 at 300 IU/ml for 5 days

• Antibody-Mediated Modulation:

  • Administration of anti-BTLA antibodies (e.g., 6F7) at specific doses and schedules

  • In NZB/W mice studies, 3 mg/kg twice weekly for ten weeks was used

• Adoptive Transfer Models:

  • TCR-transgenic T cells (e.g., OT-1) from BTLA-KO mice can be transferred into recipient mice

  • This allows for antigen-specific studies of BTLA function

• Patient-Derived Xenograft (PDX) Models:

  • Immunodeficient NSG mice can be used to study human BTLA biology in vivo

  • Combined with adoptive transfer of BTLA-manipulated human TILs

How does BTLA interact with other co-inhibitory receptors in regulating mouse immune responses?

BTLA functions within a complex network of co-inhibitory receptors that collectively regulate immune responses:

• Complementary Inhibitory Pathways: BTLA likely works alongside other inhibitory receptors like PD-1, CTLA-4, LAG-3, and TIM-3 to maintain immune homeostasis.

• Cell Type-Specific Regulation: While BTLA regulates both B and T cells, it may have unique roles that complement other co-inhibitory receptors with more restricted expression patterns.

• Disease Context Specificity: In lupus models, BTLA dysfunction occurs alongside alterations in other regulatory pathways, suggesting a complex interplay rather than isolated dysfunction.

• Therapeutic Implications: Understanding the relationship between BTLA and other co-inhibitory receptors is essential for developing combination therapies for autoimmune diseases or cancer.

What mouse strains are optimal for BTLA research in different disease contexts?

The choice of mouse strain significantly impacts BTLA research outcomes:

The NZB/W mouse model is considered particularly suitable for studying lupus pathogenesis and therapeutic strategies due to its close resemblance to human SLE as confirmed by multi-omics analysis .

What methodological considerations are important when studying BTLA in aging mice?

When studying BTLA in aging mice, researchers should consider:

• Age-Related Changes: BTLA expression and function change with age, particularly in disease models. Studies should compare young (10-12 weeks) with old mice (>35 weeks) to capture these differences .

• Subset Analysis: The shift from naive to memory T cells with age significantly impacts BTLA functionality. Cell subset-specific analysis is essential rather than examining total populations .

• Functional Assays: Memory CD4+ T cells from old NZB/W mice are refractory to in vitro activation, complicating functional studies. Alternative approaches may be needed for these populations .

• Control Selection: Age-matched controls are critical, as even healthy mice show immunological changes with aging.

• Disease Stage Assessment: In lupus models, correlate BTLA alterations with disease markers (e.g., proteinuria) rather than just chronological age .

How might BTLA targeting be combined with other immunotherapies in mouse models?

Potential combination strategies involving BTLA targeting include:

• Multi-Checkpoint Inhibition: Combining BTLA modulation with targeting of other inhibitory receptors (PD-1, CTLA-4) could provide synergistic effects in cancer or autoimmune models.

• Cytokine Therapy: Pairing BTLA-targeting antibodies with cytokine treatments could enhance therapeutic efficacy in autoimmune disease models.

• Cell-Based Therapies: Engineering adoptively transferred T cells (like CAR-T) with modified BTLA signaling domains could improve their persistence and function in solid tumor models.

• Targeting Multiple Cell Types: Since BTLA affects both B and T cells, combination approaches targeting cell type-specific pathways alongside BTLA could provide more comprehensive disease control.

• Sequential Therapy: Administering BTLA-targeting agents at specific timepoints relative to other immunotherapies might maximize beneficial effects while minimizing adverse events.

What are promising approaches for translating mouse BTLA findings to human immunotherapy?

Translational strategies for moving from mouse BTLA research to human applications:

• Comparative Expression Analysis: Detailed comparison of BTLA expression patterns between mouse models and human patient samples can identify conserved features as therapeutic targets.

• PDX Models: Patient-derived xenograft models using NSG mice can bridge the gap between mouse findings and human applications by studying human BTLA in vivo .

• Humanized Mouse Models: Mice reconstituted with human immune systems allow testing of human-specific BTLA-targeting therapies.

• Cross-Species Functional Validation: Testing whether BTLA-targeting approaches that work in mice (e.g., the 6F7 antibody) have similar effects on human cells in vitro.

• Biomarker Development: Identifying BTLA-related biomarkers in mouse models that predict therapeutic response, then validating these in human samples.