Recombinant Human B- and T-lymphocyte attenuator (BTLA), partial

What is BTLA and what is its primary function in the immune system?

BTLA is an inhibitory immune checkpoint receptor belonging to the CD28 family that plays a crucial role in maintaining immune homeostasis. When BTLA engages with its ligand HVEM, it induces phosphorylation of immunoreceptor tyrosine-based inhibitory motifs (ITIMs) in its cytoplasmic tail, leading to recruitment of tyrosine phosphatases SHP1 and SHP2 . These phosphatases dephosphorylate key signaling molecules in T cell activation pathways, resulting in suppression of T cell proliferation, cytokine production, and effector functions . BTLA shares functional and structural similarity with other checkpoint receptors such as CTLA-4 and PD-1, delivering inhibitory signals that reduce cellular activation and growth .

How does BTLA interact with HVEM at the molecular level?

The crystal structure of the BTLA/HVEM complex (Protein Data Bank ID 2AW2) reveals that BTLA interacts primarily with the first cysteine-rich domain (CRD1) of HVEM . The binding interface involves a β-hairpin structure formed by residues 23-39 of HVEM's CRD1 domain, which creates an anti-parallel inter-molecular β-sheet with the G° strand of BTLA . Key residues in this interaction include Tyr23 and Val36 of HVEM, which serve as hot-spots for binding . The stability of this interaction is maintained through multiple hydrogen bonds that remain largely preserved during molecular dynamics simulations . The HVEM CRD1 domain contains four short β-strands connected by three disulfide bonds formed between cysteine residues 4-15, 16-29, and 19-37, contributing to the structural stability of the interaction interface .

What cell types express BTLA and how is its expression regulated?

BTLA is predominantly expressed on various immune cells, including:

In cancer contexts such as hepatocellular carcinoma (HCC), BTLA expression is elevated on tumor-infiltrating T cells, with over 85% of BTLA+ CD4+ T cells co-expressing PD-1 . The regulation of BTLA expression involves both transcriptional and post-translational mechanisms influenced by cytokine signaling, T cell receptor engagement, and interactions with other immune checkpoint pathways .

What are the optimal methods to quantify BTLA expression on immune cells?

Several complementary methodological approaches can be employed to accurately quantify BTLA expression:

• Flow Cytometry: The gold standard approach involves fluorescence-activated cell sorting (FACS) using fluorochrome-conjugated anti-BTLA monoclonal antibodies. Multi-parameter flow cytometry allows simultaneous detection of BTLA along with other surface markers to identify specific cell subsets .

• Magnetic Cell Separation (MACS): For isolation of BTLA-expressing cells, magnetic cell separation using anti-BTLA antibodies coupled to magnetic beads can be employed, followed by flow cytometric analysis to confirm purity .

• Immunohistochemistry/Immunofluorescence: These techniques can assess BTLA expression in tissue sections, providing spatial context for expression patterns.

• Quantitative PCR: For analysis of BTLA mRNA expression, qPCR can be used, although this does not provide information about protein localization or expression on specific cell subsets unless combined with cell sorting.

When analyzing BTLA expression in disease contexts, it is essential to include appropriate controls and to consider co-expression with other immune checkpoints like PD-1, as co-expression patterns can provide valuable insights into the functional state of immune cells .

How can recombinant BTLA proteins be used in experimental settings?

Recombinant BTLA proteins serve as valuable tools in various experimental applications:

When working with recombinant BTLA proteins, researchers should consider the presence of tags (e.g., His-tag), the expression system used for production, and potential post-translational modifications that might affect function or interaction properties .

What experimental approaches can be used to study BTLA-HVEM interactions?

Multiple experimental approaches provide complementary insights into BTLA-HVEM interactions:

• Molecular Modeling and Simulations: Using the crystal structure of BTLA/HVEM complex (PDB ID 2AW2), molecular dynamics simulations with GROMACS and the CHARMM22 force field can assess interaction stability and dynamics over time .

• ELISA-Based Binding Assays: Direct binding assays can be performed by coating plates with recombinant BTLA-Fc protein (400 ng/well) and then adding titrated amounts of HVEM-Fc protein. The interaction can be detected using appropriately labeled secondary antibodies .

• Competition Assays: To identify regions important for binding or to screen potential inhibitors, competition assays can test peptides or small molecules for their ability to disrupt the BTLA-HVEM interaction .

• Ellman's Assay: For investigating the role of free thiols in the interaction, Ellman's assay using DTNB can measure free thiol content in protein samples, providing insights into the biochemical nature of the interaction .

• Surface Plasmon Resonance (SPR): SPR enables real-time kinetic analysis of protein-protein interactions, determining association and dissociation rates as well as binding affinities.

These approaches provide structural, biochemical, and functional information about BTLA-HVEM interactions, essential for comprehensive understanding of this important immune regulatory axis .

How does BTLA expression correlate with disease progression in cancer?

BTLA expression demonstrates significant correlations with disease progression and outcomes across several cancer types:

The relationship between BTLA expression and disease progression appears related to its role in promoting T cell exhaustion and impairing antitumor immunity . The co-expression of BTLA with other inhibitory checkpoints like PD-1 further contributes to the functional impairment of tumor-infiltrating lymphocytes, facilitating immune evasion and tumor progression .

What are the differences between BTLA and PD-1 in regulating T cell responses?

BTLA and PD-1 exhibit important differences in their mechanisms of action and effects on T cell responses:

• Signaling Mechanisms: While both BTLA and PD-1 recruit SHP1 and SHP2 phosphatases, they affect downstream signaling pathways differently. BTLA potently inhibits the phosphorylation of both TCR (CD3ζ) and CD28, whereas PD-1 may have more selective effects on specific signaling pathways .

• Expression Patterns: In hepatocellular carcinoma, about 83 ± 6.5% of BTLA-expressing tumor CD4+ T cells are PD-1+, whereas only 54 ± 7.9% of PD-1-expressing tumor CD4+ T cells are BTLA+, suggesting they mark partially overlapping but distinct T cell populations .

• Functional Effects: BTLA engagement results in a more robust inhibition of IL-2 production and CD28 phosphorylation compared to PD-1 engagement in some experimental systems . BTLA can suppress T cell signaling through mechanisms independent of both SHP1 and SHP2 .

• Response to Blockade: The effects of blocking BTLA versus PD-1 vary depending on the disease context. In some cases, dual BTLA/PD-1 blockade shows enhanced efficacy compared to monotherapies, suggesting non-redundant roles .

• Ligand Interactions: Unlike PD-1, which interacts with PD-L1 and PD-L2, BTLA interacts with HVEM, a member of the TNF receptor superfamily. This cross-family interaction adds complexity not present in the PD-1 pathway .

These differences are crucial for developing targeted therapeutic approaches and predicting the effects of blocking these pathways in different disease contexts .

How does BTLA contribute to immune cell exhaustion in chronic diseases?

BTLA contributes to immune cell exhaustion through several interconnected mechanisms:

• Sustained Inhibitory Signaling: Persistent engagement of BTLA with HVEM in chronic disease settings leads to continuous inhibitory signaling, suppressing T cell activation, proliferation, and effector functions over time .

• Altered Cytokine Production: BTLA activation significantly reduces the production of key cytokines required for effective immune responses. In CLL, BTLA activation leads to decreased percentages of:

  • IL-2+ CD3+ T cells: 33.81 ± 5.57% vs. 21.71 ± 6.93% (p=0.01)

  • IL-2+ CD4+ T cells: 37.03 ± 5.52% vs. 23.87 ± 8.05% (p=0.01)

  • IL-2+ CD8+ T cells: 18.05 ± 4.02% vs. 10.35 ± 3.05% (p=0.01)

  • Similar reductions in IFN-γ across all T cell subsets

• Impact on TCR Signaling Pathways: BTLA engagement suppresses key T cell signaling pathways, including MAPK, NF-κB, and NFAT activation, which are essential for T cell function . This broad inhibition of signaling contributes to the progressive functional impairment characteristic of exhausted T cells.

• Co-expression with Other Inhibitory Receptors: In chronic diseases, BTLA is often co-expressed with other inhibitory receptors like PD-1, creating a multi-layered suppressive environment that enhances T cell exhaustion .

BTLA blockade has shown promise in reversing some aspects of T cell exhaustion. For example, BTLA blockade enhances IFN-γ production, particularly in CD8+ T cells, and can boost cytotoxic responses against tumor cells, suggesting that BTLA contributes to the reversible component of T cell exhaustion .

What is the relationship between BTLA expression and response to immunotherapy?

The relationship between BTLA expression and immunotherapy response is complex and context-dependent:

• Predictive Biomarker Potential: BTLA expression on T cells may serve as a predictive biomarker for response to immunotherapy. In some cancers, high BTLA expression is associated with poor outcomes and may indicate patients who could benefit from targeted approaches .

• Impact on Checkpoint Inhibitor Efficacy: BTLA on tumor-infiltrating lymphocytes may limit the efficacy of other checkpoint inhibitors, such as PD-1/PD-L1 blockade. Studies have shown that BTLA+ cells identify highly dysfunctional PD-1-expressing CD4+ T cell subsets in HCC .

• Differential Effects of Blockade: BTLA blockade enhances IFN-γ production by T cells, particularly CD8+ T cells, but may have limited effects on IL-2 production. This suggests that BTLA blockade may selectively enhance certain aspects of T cell function .

• Combination Approaches: Emerging evidence suggests that targeting BTLA in combination with other immunotherapeutic approaches provides enhanced efficacy. In CLL models, the combination of BTLA blockade with bispecific anti-CD3/anti-CD19 antibodies significantly boosted CD8+ T cell anti-leukemic activity .

• Emerging Clinical Data: Clinical trials with novel anti-BTLA monoclonal blocking antibodies, such as icatolimab, are showing promising preliminary results in patients with advanced solid tumors .

Understanding this complex interplay is crucial for optimizing immunotherapeutic approaches and identifying patients most likely to benefit from BTLA-targeted interventions .

What approaches can be used to develop therapeutics targeting BTLA-HVEM interactions?

Several sophisticated approaches can be employed to develop therapeutics targeting BTLA-HVEM interactions:

• Monoclonal Antibodies: Developing antibodies that block the BTLA-HVEM interaction is a primary approach. Icatolimab, a first-in-class anti-BTLA monoclonal blocking antibody, has shown promising preliminary results in clinical trials for advanced solid tumors .

• Engineered HVEM Variants: Computational design methods like ProtLID (Protein Ligand Interface Design) can generate residue-based pharmacophores over the binding interfaces of HVEM. Single mutations like H86I and D100R on HVEM reduce HVEM binding to LIGHT, while double mutants like D100R with M103K achieve BTLA-selective binding .

• Peptide Inhibitors: Peptides that mimic key interaction regions can provide competitive inhibitors of BTLA-HVEM binding. Molecular dynamics simulations show that the HVEM(23-39) fragment can stably interact with BTLA, suggesting it as a potential starting point for peptide inhibitor design .

• Structural-Based Design: The crystal structure of BTLA/HVEM complex (PDB ID 2AW2) provides a foundation for structure-based drug design approaches to develop molecules that interfere with this interaction .

• Bispecific Antibodies: Developing bispecific antibodies that simultaneously target BTLA and another relevant molecule can provide more specific modulation of immune responses in the tumor microenvironment.

These approaches provide various strategies for therapeutic intervention in the BTLA-HVEM pathway, each with distinct advantages and challenges in terms of specificity, efficacy, and clinical translation .

How does BTLA blockade affect different immune cell subsets in various disease contexts?

BTLA blockade has differential effects on immune cell subsets depending on the disease context:

In cancer models like CLL, BTLA blockade enhances CD8+ T cell cytotoxicity against leukemic cells, particularly when combined with bispecific anti-CD3/anti-CD19 antibodies . In contrast, in sepsis models, anti-BTLA antibody treatment increased cytokine/chemokine production and inflammatory cell recruitment, exacerbating organ injury and increasing mortality .

In renal transplantation, BTLA plays a protective role by suppressing acute rejection. Overexpression of BTLA in rat models significantly inhibited acute rejection and prolonged allograft survival by suppressing IL-2 and IFN-γ production while increasing IL-4 and IL-10 production .

These diverse effects highlight the importance of understanding BTLA's specific role in different disease contexts when developing therapeutic strategies targeting this pathway .

What are potential combination approaches for targeting BTLA along with other immune checkpoints?

Optimizing combination approaches targeting BTLA and other immune checkpoints requires several strategic considerations:

• Checkpoint Co-expression Analysis: Detailed profiling of BTLA co-expression with other immune checkpoints guides rational combination strategies. In HCC, BTLA+ cells represent a subset of PD-1+ CD4+ T cells with heightened dysfunction, suggesting potential benefits of dual targeting .

• BTLA and PD-1 Dual Blockade: Studies suggest that dual BTLA/PD-1 blockade results in heightened IFN-γ levels and improved outcomes compared to monotherapies in some cancer models .

• Integration with Targeted Therapies: In CLL, the combination of BTLA blockade with ibrutinib (a BTK inhibitor) significantly increased leukemic cell depletion without affecting T cell numbers, suggesting beneficial interaction between these approaches .

• Biomarker-Guided Selection: Patients with high co-expression of BTLA and PD-1 on tumor-infiltrating lymphocytes might be more suitable candidates for dual blockade approaches .

• Sequence Optimization: Determining whether sequential or simultaneous blockade of multiple checkpoints provides optimal efficacy is crucial, as priming with one checkpoint inhibitor may alter the expression or function of others.

By addressing these considerations, combination approaches targeting BTLA along with other immune checkpoints can potentially extend immunotherapy benefits to a broader range of patients .