Recombinant Mouse Interleukin-15 receptor subunit alpha (Il15ra), partial (Active)

What is mouse IL-15Rα and how does it differ from human IL-15Rα?

Mouse interleukin-15 receptor subunit alpha (Il15ra) functions as a high-affinity receptor for interleukin-15. It associates as a heterotrimer with the IL-2 receptor beta and gamma subunits (Common gamma chain, or γc) to initiate signal transduction. Mouse Il15ra is expressed in various T and B cells and non-lymphoid cells . Human IL-15Rα shares approximately 45% amino acid sequence homology with the mouse form of the receptor . This difference in sequence homology is significant but does not prevent functional cross-reactivity, as human IL-15Rα protein can bind to mouse IL-15 . When designing cross-species experiments, researchers should account for these structural differences while leveraging the functional conservation.

What are the known isoforms of mouse IL-15Rα?

Eight isoforms of IL-15Rα mRNA have been identified, resulting from alternative splicing events involving different exons . These splice variants likely contribute to the diverse functional roles of IL-15Rα across different tissue types and physiological contexts. When designing experiments targeting IL-15Rα, researchers should consider which isoforms are relevant to their specific research questions and select appropriate detection methods that can distinguish between these variants.

What is trans-presentation and how does it relate to IL-15Rα function?

Trans-presentation is a specialized mechanism by which IL-15 delivers its signal. In this process, IL-15 expressed at the surface of presenting cells via the membrane-bound IL-15Rα chain is presented during interaction with responding cells that express the transducing receptor . The IL-15 and IL-15Rα preassociate within presenting cells prior to emerging to the cell surface . This mechanism allows for controlled delivery of IL-15 signals to specific target cells, providing spatial and temporal regulation of immune responses. When designing experiments to study IL-15 signaling, researchers should account for this trans-presentation process, particularly when co-culture systems are employed.

Can IL-15Rα signal independently of IL-15?

Interestingly, IL-15Rα is competent for signal transduction by itself . Furthermore, studies have shown that IL-15Rα protein can function as an adjuvant with a limited immune expansion phenotype even in the absence of IL-15 . In IL-15 knockout mice, which lack any endogenous IL-15, immunization with plasmid-encoded IL-15Rα (pIL-15Rα) still enhanced cellular immune responses compared to antigen alone . This suggests that IL-15Rα possesses intrinsic signaling capabilities independent of its canonical ligand, which could be exploited for immunomodulatory approaches.

What happens to the IL-15/IL-15Rα complex after trans-presentation?

After trans-presentation, the IL-15/IL-15Rα complex can be cleaved from the surface of presenting cells. Research has identified an unprecedented cytokine pathway in which the IL-15/IL-15Rα complex cleaved from presenting cells allows responding cells to internalize, store, and use the complex for their own proliferation and survival . This mechanism provides an additional layer of regulation in IL-15 signaling, allowing for sustained effects even after the initial cell-cell interaction has ceased. Researchers should consider tracking the fate of these complexes when studying long-term effects of IL-15 signaling.

How can recombinant IL-15Rα be used to study IL-15 signaling in vitro?

Recombinant IL-15Rα can be used to create stable transfected cell lines (such as HEK-293) that express membrane-bound IL-15Rα. These cells can be cultured with IL-15 overnight to allow IL-15 binding to IL-15Rα and recycling of the complex to the cell surface . After washing to remove soluble IL-15, these cells can be used in co-culture experiments with responding cells to study trans-presentation mechanisms. Additionally, researchers can create fusion molecules comprising the IL-15Rα chain covalently linked to IL-15 to mimic the preassociation of IL-15 and IL-15Rα, allowing controlled study of trans-presentation dynamics .

What methods can be used to detect IL-15Rα expression and IL-15 binding?

Several techniques can be employed to detect IL-15Rα expression and IL-15 binding:

• Flow cytometry: Can detect surface IL-15Rα expression and IL-15 binding to cells

• Intracellular staining: Allows detection of internalized IL-15/IL-15Rα complexes

• ELISA: Can be used to measure IL-15 in cell lysates and supernatants; requires cell lysis in appropriate buffer (e.g., 25 mmol/L Tris-HCl pH 7.4, 150 mmol/L NaCl, 1% Triton X-100, and 5% glycerol)

• RT-PCR: Detects IL-15Rα mRNA expression and can distinguish between different isoforms

• Immunoprecipitation: Can be used to study IL-15/IL-15Rα interactions, as demonstrated by studies using 35S-radiolabeled human IL-15Rα protein incubated with murine IL-15

When selecting detection methods, researchers should consider the sensitivity requirements and whether they need to distinguish between surface-bound and intracellular forms of the receptor.

How can I assess the functional activity of recombinant IL-15Rα in immunological assays?

Functional activity of recombinant IL-15Rα can be assessed through several approaches:

• Cell proliferation assays: Measure the effect of IL-15Rα (alone or in complex with IL-15) on proliferation of responsive cell lines

• IFN-γ secretion: Quantify IFN-γ-secreting cells using ELISPOT assays after stimulation with IL-15Rα, as shown in studies where pIL-15Rα enhanced antigen-specific IFN-γ secretion in a dose-dependent manner

• Colony formation assays: Assess the impact of IL-15Rα manipulation on adherent cell colony formation over extended periods (e.g., 15 days)

• Knockdown/silencing experiments: Use RNAi to silence IL-15Rα in cell lines expressing either high or low levels of IL-15Rα to observe functional effects

• In vivo immunization studies: Evaluate the adjuvant effect of IL-15Rα by co-administering it with antigenic constructs and measuring immune responses

These assays provide complementary information about the biological activity of IL-15Rα in different experimental contexts.

What is the role of IL-15Rα in cancer biology and how can recombinant IL-15Rα be used in cancer research?

IL-15Rα expression varies across cancer types and subtypes. Studies have shown significant differences in IL15RA expression between "basal" versus "luminal" breast cancer cell lines . Functionally, silencing IL15RA by RNAi significantly impairs growth in high IL15RA–expressing cancer cell lines (such as MDA-MB-231) but not in low-expressing lines (such as SKBR3) . Additionally, IL15RA knockdown reduces adherent cell colony formation in high-expressing cell lines .

Researchers can use recombinant IL-15Rα in cancer studies to:

• Investigate differential effects based on baseline IL-15Rα expression

• Explore combination approaches with IL-15 or other immunomodulatory agents

• Develop targeting strategies that exploit IL-15Rα expression patterns

• Study the impact of IL-15Rα manipulation on tumor microenvironment

How does the IL-15/IL-15Rα complex (IL-15Cx) affect immune responses in inflammatory conditions?

These findings suggest that IL-15Cx has context-dependent effects on immune responses, which researchers should consider when developing IL-15-based therapeutic approaches for inflammatory conditions.

What strategies exist for targeting IL-15Rα to modulate inflammation?

One approach involves generating IL-15-derived molecules designed to selectively inhibit the action of IL-15 . A strategy called NANTIL-15 (New ANTagonist of IL-15) works by retaining the binding to IL-15Rα while preventing recruitment of IL-2Rβ, effectively blocking IL-15's signaling through the trimeric IL-15Rα/IL-2Rβ/γc receptor without affecting signaling through the dimeric IL-2Rβ/γc receptor .

This approach targets specific residues (N65 and L69) that are crucial for the binding of IL-15 to IL-2Rβ . By modifying these residues, researchers can create selective IL-15 antagonists that may offer therapeutic potential in inflammatory conditions where IL-15 signaling contributes to pathology.

What are the key considerations when designing experiments with mouse and human IL-15Rα components?

When working with mouse and human IL-15Rα components, researchers should consider:

• Cross-species reactivity: Human IL-15Rα can bind to mouse IL-15, as demonstrated by studies showing that 35S-radiolabeled human IL-15Rα protein incubated with murine IL-15 can be immunoprecipitated with an anti-mouse IL-15 antibody

• Species-specific differences: Mouse IL-15ra shares only 45% amino acid sequence homology with the human form

• Isoform selection: Eight isoforms of IL-15Rα mRNA have been identified, so researchers should consider which specific isoforms are relevant to their research question

• Fusion tags: Commercial recombinant proteins may contain fusion tags (e.g., C-Fc tag) that could affect certain experimental readouts

• Binding affinities: IL-15Rα binds IL-15 with high affinity (Kd=10–80 pM), which affects experimental design for binding and competition assays

What controls should be included when studying IL-15Rα function independently of IL-15?

When investigating IL-15Rα function independently of IL-15, several controls are essential:

• IL-15 knockout models: Studies in IL-15 knockout mice can confirm IL-15-independent effects of IL-15Rα

• Split delivery controls: When studying complex formation, separate delivery of IL-15 and IL-15Rα components (e.g., injecting plasmids in separate anatomical locations) can help determine whether local complex formation is necessary

• Dose dependency: Testing increasing doses of IL-15Rα can establish whether effects occur in a dose-dependent manner

• Anti-IL-15Rα antibody controls: Verifying the absence of anti-IL-15Rα antibody responses in experimental subjects rules out potential confounding immunological reactions

• Verification of endogenous IL-15 levels: Measuring baseline IL-15 in experimental systems helps interpret IL-15Rα-mediated effects

These controls help distinguish IL-15-dependent from IL-15-independent effects of IL-15Rα.

What technical challenges might arise when working with recombinant IL-15Rα in experimental systems?

Researchers working with recombinant IL-15Rα may encounter several technical challenges:

• Solubility and stability: Recombinant proteins may have limited solubility or stability under certain experimental conditions

• Cleavage of membrane-bound IL-15Rα: IL-15Rα can be cleaved from the surface of presenting cells, potentially complicating interpretation of experimental results

• Complex formation dynamics: The kinetics of IL-15/IL-15Rα complex formation and dissociation can affect experimental outcomes

• Detection sensitivity: Given the high-affinity binding and potentially low expression levels, sensitive detection methods may be required

• Background IL-15 levels: Endogenous IL-15 in experimental systems can confound interpretation of IL-15Rα-specific effects

• Isoform heterogeneity: Multiple IL-15Rα isoforms may contribute differently to observed effects

• Cross-reactivity considerations: When using components from different species, unexpected cross-reactivity may occur

Addressing these challenges requires careful experimental design, appropriate controls, and validation using complementary methodological approaches.