AITRL Human, T7

What is AITRL Human T7 Tag and what are its key structural characteristics?

AITRL Human Recombinant produced in E.Coli is a single, non-glycosylated polypeptide chain containing 134 amino acids (residues 81-199) with a molecular mass of approximately 15kDa . The protein features a T7 tag which facilitates detection and purification in laboratory settings. Also known as Glucocorticoid-Induced TNF-Related Ligand (GITRL), AITRL belongs to the tumor necrosis factor superfamily (TNFSF18) .

The recombinant protein's full sequence is: "MASMTGGQQM GRGSHMAKFG PLPSKWQMAS SEPPCVNKVS DWKLEILQNG LYLIYGQVAP NANYNDVAPF EVRLYKNKDM IQTLTNKSKI QNVGGTYELH VGDTIDLIFN SEHQVLKNNT YWGIILLANP QFIS" . This sequence includes the T7 tag portion at the N-terminus followed by the human AITRL sequence. As a Type II single transmembrane protein, AITRL exhibits relatively low conservation within its extracellular domain compared to other TNFSF members .

What are the expression patterns and biological functions of AITRL?

AITRL demonstrates tissue-specific expression with high levels detected in the small intestine, ovary, testis, and kidney . It is expressed on several immune cell types, including macrophages, immature and mature dendritic cells, and B cells . Notably, endothelial cells significantly upregulate AITRL expression following stimulation with lipopolysaccharides, IFN-alpha, or IFN-beta .

Functionally, AITRL plays critical roles in immune regulation through binding to its receptor AITR (TNFRSF18/GITR), which is expressed on T lymphocytes, natural killer (NK) cells, and antigen-presenting cells . This interaction facilitates several important processes:

• Modulation of T-lymphocyte survival in peripheral tissues

• Enhancement of TCR-induced T cell proliferation and cytokine production

• Protection of T cells and NK cells from apoptosis

• Increased resistance to tumors and viral infections

• Involvement in autoimmune and inflammatory processes

Additionally, AITRL (also referred to as Osteostat) acts as a regulator of bone physiology by inhibiting osteoclast differentiation from monocytic precursor cells .

How does AITRL inhibit osteoclastogenesis?

AITRL (Osteostat) specifically inhibits the differentiation of osteoclasts from monocytic precursor cells through a well-defined mechanism . It suppresses the early stage of osteoclastogenesis by inhibiting macrophage colony-stimulating factor-induced receptor activator of NF-kappaB (RANK) expression in osteoclast precursor cells .

This inhibitory effect demonstrates remarkable specificity, as AITRL does not inhibit lipopolysaccharide-induced RANK expression in monocytes and dendritic cells, nor does it affect activation-induced RANK expression in T cells . This selective action positions AITRL as a novel regulator of osteoclast generation and highlights the significant role played by the endothelium in bone physiology .

Researchers investigating bone metabolism disorders or developing treatments for conditions like osteoporosis may find AITRL particularly relevant as a potential therapeutic target or research tool.

What methodological approaches are recommended for studying AITRL-AITR interactions in experimental systems?

When investigating AITRL-AITR interactions, researchers should consider multiple complementary approaches to obtain robust data:

• Recombinant protein assays: Utilize purified AITRL Human T7 Tag protein (>90% purity by SDS-PAGE) in binding studies with AITR-expressing cells or recombinant AITR protein. Fluorescently labeled AITRL can be employed to visualize binding, as demonstrated in studies where AITRL-Fc was used to detect upregulated GITR on activated T cells .

• Cell-based functional assays: Establish co-culture systems with AITRL-expressing cells (such as activated dendritic cells) and AITR-expressing cells (like T cells or NK cells). Measure functional outcomes including proliferation, cytokine production, and apoptosis resistance .

• Expression induction systems: To study native AITRL expression, stimulate human microvascular endothelial cells with IFN-alpha or IFN-beta, which significantly upregulate AITRL . Alternatively, use LPS to induce AITRL expression on B cells in vitro .

• Single-cell perturbation approaches: Apply techniques like Perturb-seq, which combines droplet-based single-cell transcriptomics with CRISPR-Cas based perturbations to analyze transcriptional responses to AITRL pathway manipulation . This approach allows for the resolution of novel phenotypes and cell-to-cell variability in response.

When validating binding, researchers should follow protocols similar to those described in the literature where AITRL-Fc binding to peptide-specific CD4+6.5+ T cells was assessed following activation with cognate peptide .

How can AITRL be integrated into combinatorial perturbation experiments to elucidate immune regulatory networks?

AITRL represents an excellent candidate for inclusion in combinatorial perturbation experiments aimed at understanding complex immune cell circuits. Advanced methodological approaches include:

• Multi-component perturbation analysis: Implement the Perturb-seq methodology, which enables massively parallel analysis of genetic perturbations at the single-cell level . For AITRL studies, this would involve:

  • Creating a library of sgRNAs targeting AITRL and other immune regulatory genes

  • Introducing multiple sgRNAs into single cells to achieve combinatorial perturbations

  • Analyzing transcriptional responses through single-cell RNA sequencing

  • Applying computational frameworks like MIMOSCA (Multi-Input Multi-Output Single Cell Analysis) to identify regulatory effects

• Epistatic mapping: Design perturbation experiments that target AITRL alongside its upstream regulators or downstream effectors to build epistatic maps of AITRL-mediated signaling . This approach can reveal genetic interactions including synergistic, buffering, and dominant interactions that cannot be predicted from individual perturbations alone.

• Cell-type specific analyses: Leverage the single-cell resolution of these methods to identify differential responses to AITRL perturbation across immune cell subpopulations . This can uncover bifurcations in cellular behavior and cell type-specific regulatory mechanisms.

This combinatorial approach offers a 10-fold improvement in cost compared to traditional methods for obtaining perturbation transcription profiles and enables systematic dissection of epistatic effects using RNA transcription as a phenotype .

What considerations are important when using AITRL in T regulatory cell (T-reg) research?

Researchers studying T regulatory cells should carefully consider the complex role of the AITRL-AITR pathway in modulating T-reg function:

• Differential impact on effector vs. regulatory T cells: Engagement of AITR (GITR) on T cells by AITRL on APCs or by agonistic antibodies has distinct effects on different T cell populations. It co-stimulates effector cells and may render them resistant to T-reg suppression, while simultaneously diminishing the suppressive capacity of T-regs themselves .

• Paradoxical effects on T-reg proliferation: Despite reducing suppressive function, GITR activation promotes T-reg proliferation in response to TCR stimulation . This apparent contradiction requires careful experimental design to distinguish between numeric expansion and functional suppression.

• Role in transplantation tolerance: GITR blockade has been shown to facilitate T-reg mediated allograft survival, indicating that inhibiting this pathway may enhance T-reg function in transplantation settings . Researchers should account for this when designing experiments related to transplantation or tolerance induction.

• Context-dependent activation: AITRL expression is upregulated on B cells after LPS treatment and on antigen-presenting cells in draining lymph nodes following viral infection . These context-dependent expression patterns suggest that the AITRL-AITR pathway serves as a mechanism by which innate immune stimuli can modulate adaptive immune responses through effects on T-regs.

• Experimental validation: When studying AITRL-AITR interactions in T-reg contexts, researchers should include appropriate controls and validation steps, such as confirming binding of AITRL to activated T cells as demonstrated in previous studies .

How does AITRL expression change under different inflammatory conditions and what are the regulatory mechanisms?

AITRL expression demonstrates significant modulation under various inflammatory conditions through multiple regulatory mechanisms:

• Type I interferon regulation: Human microvascular endothelial cells show baseline AITRL expression that is dramatically upregulated in response to IFN-alpha and IFN-beta . This suggests AITRL may play a role in antiviral immune responses.

• Bacterial component induction: Lipopolysaccharide (LPS) stimulation induces AITRL expression in endothelial cells and B cells . This represents a pathway through which bacterial infection can enhance adaptive immune responses.

• Viral infection response: Antigen-presenting cells in draining lymph nodes upregulate AITRL following herpes simplex virus exposure in vivo . This pattern of expression allows AITRL to function as a bridge between innate viral sensing and adaptive immune activation.

The dynamic regulation of AITRL underscores its importance in coordinating appropriate immune responses to different inflammatory triggers. Researchers should consider these expression patterns when designing experiments to study AITRL function in specific disease contexts or when developing therapeutic strategies targeting this pathway.

What validation methods should be employed when working with AITRL Human T7 Tag protein?

To ensure experimental reliability when working with AITRL Human T7 Tag protein, researchers should implement a multi-faceted validation approach:

• Purity assessment: Confirm protein purity via SDS-PAGE, with acceptable purity standards exceeding 90% .

• Binding validation: Verify functional binding to the GITR receptor using flow cytometry or other binding assays. This can be demonstrated by incubating AITRL-Fc with activated T cells that upregulate GITR expression, such as peptide-specific CD4+6.5+ T cells .

• Sequence verification: Confirm the protein sequence through mass spectrometry or N-terminal sequencing, comparing results to the expected sequence: "MASMTGGQQM GRGSHMAKFG PLPSKWQMAS SEPPCVNKVS DWKLEILQNG LYLIYGQVAP NANYNDVAPF EVRLYKNKDM IQTLTNKSKI QNVGGTYELH VGDTIDLIFN SEHQVLKNNT YWGIILLANP QFIS" .

• Functional testing: Assess biological activity through relevant functional assays, such as T cell proliferation assays, cytokine production measurement, or osteoclast differentiation inhibition assays .

• Specificity controls: Include appropriate negative controls such as irrelevant proteins with similar tags and blocking antibodies to confirm the specificity of observed effects.

These validation steps are essential for ensuring that experimental results obtained with AITRL Human T7 Tag protein are reliable and reproducible across different research settings.

How can researchers differentiate between AITRL signaling and other TNF superfamily members in complex experimental systems?

Distinguishing AITRL signaling from other TNF superfamily members requires specialized experimental approaches:

• Targeted genetic perturbation: Utilize CRISPR-Cas based perturbations specifically targeting AITRL or its receptor AITR. The Perturb-seq approach combining these perturbations with single-cell transcriptomics allows for precise identification of AITRL-specific effects even in complex cellular systems .

• Specific antibody blocking: Employ highly specific antibodies that recognize unique epitopes on AITRL that are not conserved among other TNFSF members. This is particularly important since AITRL shares low conservation within the extracellular domain with other TNFSF members .

• Receptor-specific readouts: Focus on downstream effects that are uniquely associated with AITR activation, such as specific transcriptional signatures or phosphorylation events.

• Comparative analysis: Perform side-by-side experiments with multiple TNFSF members to identify distinct patterns of response that characterize AITRL signaling versus other family members.

• Cell type specificity: Leverage the known expression patterns of AITRL on specific cell types (macrophages, dendritic cells, B cells) to design experiments that can distinguish AITRL-mediated effects from those of other TNFSF members with different cellular distribution patterns.

These approaches can be combined within experimental frameworks that allow for systematic dissection of cellular responses, such as those described for analyzing complex responses like the unfolded protein response .

What challenges exist in targeting the AITRL-AITR pathway for immunotherapeutic applications?

Developing therapeutics targeting the AITRL-AITR pathway presents several significant challenges that researchers must address:

• Dual effects on effector and regulatory T cells: The AITRL-AITR pathway simultaneously co-stimulates effector T cells while diminishing regulatory T cell suppressive function . This dual effect creates complexity in predicting the net outcome of pathway modulation in different disease contexts.

• Context-dependent expression: AITRL expression is dynamically regulated by inflammatory stimuli including type I interferons and bacterial components . This context-dependent expression must be considered when designing therapeutic interventions targeting specific disease states.

• Tissue specificity considerations: AITRL is expressed at high levels in specific tissues including the small intestine, ovary, testis, and kidney . Therapeutic modulation of this pathway may therefore have variable effects across different tissue compartments.

• Balance between beneficial and detrimental effects: While AITR activation increases resistance to tumors and viral infections (beneficial in cancer and infectious disease contexts), it is also involved in autoimmune and inflammatory processes (potentially detrimental in autoimmune conditions) . This requires careful therapeutic design to achieve the desired immunomodulatory effect while minimizing adverse outcomes.

• Transplantation implications: GITR (AITR) blockade facilitates T-reg mediated allograft survival , indicating that therapeutic approaches targeting this pathway could impact transplantation tolerance. This requires specific consideration in patients with transplanted organs.

Addressing these challenges requires sophisticated therapeutic design strategies that account for the complex biology of the AITRL-AITR pathway across different disease contexts and tissue environments.

How might AITRL's role in osteoclastogenesis be leveraged for bone-related therapeutic applications?

AITRL's inhibitory effect on osteoclastogenesis presents intriguing possibilities for therapeutic applications in bone-related disorders:

• Mechanism of action: AITRL (Osteostat) specifically suppresses the early stage of osteoclastogenesis by inhibiting macrophage colony-stimulating factor-induced receptor activator of NF-kappaB (RANK) expression in osteoclast precursor cells . This targeted mechanism offers potential advantages over broader anti-resorptive approaches.

• Therapeutic potential: As an inhibitor of osteoclast differentiation, AITRL or mimetic compounds could be developed as novel treatments for osteoporosis and other bone loss disorders. The specificity of AITRL's action—not affecting RANK expression in monocytes, dendritic cells, or T cells —suggests potential for targeted therapy with minimal off-target effects.

• Delivery considerations: Since endothelial cells naturally express AITRL, especially after stimulation with inflammatory mediators , therapeutic strategies might include approaches to enhance endogenous AITRL production in the bone microenvironment or targeted delivery of recombinant AITRL to sites of bone remodeling.

• Combination therapy potential: AITRL-based therapies might complement existing bone-directed treatments through its unique mechanism of action, potentially allowing for reduced dosing of current therapies or addressing treatment-resistant cases.

• Biomarker applications: Given its role in bone metabolism, AITRL levels might serve as a biomarker for bone remodeling activity or predictor of therapeutic response in bone-related disorders.

These therapeutic applications highlight the potential translational value of basic research into AITRL's biological functions beyond its well-established role in immune regulation.