IL 12 p35 Human

What is IL-12p35 and which cytokines incorporate this subunit?

IL-12p35 functions as a critical subunit that forms two distinct heterodimeric cytokines with opposing immunological functions. When paired with the IL-12p40 subunit, it forms IL-12, which promotes inflammatory responses. Alternatively, when paired with Ebi3 (Epstein-Barr virus-induced gene 3), it forms IL-35, which induces regulatory responses and suppresses autoimmune diseases . This dual functionality highlights how the same protein subunit can mediate opposing immunological effects depending on its heterodimeric partner .

Does IL-12p35 possess biological activity as a single-chain protein?

Research has demonstrated that IL-12p35 does indeed possess intrinsic immunoregulatory functions as a single-chain protein. Studies have shown that recombinant IL-12p35 can suppress lymphocyte proliferation in a dose-dependent manner . Furthermore, IL-12p35 induces expansion of IL-10-expressing and IL-35-expressing B cells and ameliorates autoimmune uveitis in mice by antagonizing pathogenic Th17 responses . These findings suggest that IL-12p35 has significant immunoregulatory properties independent of its heterodimeric partners .

How is the production of IL-12p35 regulated in human cells?

The production of IL-12p35 is stringently regulated through both transcriptional and post-transcriptional mechanisms. At the transcriptional level, IL-12p35 expression requires the differentiative effects of IFN-γ . Post-transcriptionally, IL-12p35 undergoes a unique processing pathway unlike most secreted proteins. The translocation of p35 preprotein into the endoplasmic reticulum (ER) is not accompanied by immediate cleavage of the signal peptide. Instead, p35 undergoes two sequential cleavages: the first occurs within the ER at the middle of the hydrophobic region of the signal peptide, and the second cleavage removes the remaining portion of the signal peptide, possibly involving a metalloprotease, concurrent with additional glycosylation and secretion . This unusual processing mechanism provides additional regulatory control over IL-12 production.

What expression systems are optimal for producing recombinant human IL-12p35?

Based on published research, insect cell expression systems have proven effective for producing recombinant IL-12p35. The methodology involves:

• Cloning IL-12p35 cDNA into an expression vector (such as pMIB) containing an amino-terminal secretion signal sequence (e.g., honeybee melittin) and a poly-histidine tag to facilitate isolation and characterization

• Driving expression using baculovirus immediate-early promoters

• Transfecting the construct into insect cells (such as High Five cells)

• Identifying stable transfectants through drug selection (e.g., Blasticidin S)

This approach yields both monomeric p35 and p35-p35 homodimeric proteins, both of which demonstrate biological activity in suppressing lymphocyte proliferation .

How can researchers assess the functional activity of recombinant IL-12p35?

Researchers can evaluate the biological activity of recombinant IL-12p35 through several complementary methodologies:

Table 1: Assays for Evaluating IL-12p35 Biological Activity

What molecular techniques can distinguish between IL-12p35 and other IL-12 family cytokines?

To differentiate IL-12p35 from heterodimeric IL-12 or IL-35, researchers can employ several molecular approaches:

• Specific antibody-based assays that recognize epitopes unique to monomeric IL-12p35

• Structure-function analysis using chimeric molecules, such as mouse-human p35 chimeras, to identify regions critical for specific functions

• Western blotting under non-reducing versus reducing conditions to distinguish between monomeric and heterodimeric forms

• Functional assays comparing activities on cells from different species, as mouse IL-12p35 functions on both mouse and human cells, while human IL-12p35 acts only on human cells

How does IL-12p35 modulate autoimmune disease progression in experimental models?

IL-12p35 has demonstrated significant therapeutic potential in multiple experimental autoimmune disease models:

Table 2: Effects of IL-12p35 in Autoimmune Disease Models

The therapeutic effects of IL-12p35 appear to be mediated through:

• Suppression of pathogenic Th17 responses

• Expansion of regulatory T and B cells

• Reduction in inflammatory myeloid cell recruitment to disease sites

• Induction of partial anergy in pathogenic T cells, diminishing their capacity to transfer disease

What are the mechanistic differences between IL-12p35 and IL-35 in immunoregulation?

Despite sharing the p35 subunit, IL-12p35 and IL-35 exhibit distinct mechanisms of immunoregulation:

Table 3: Comparative Mechanisms of IL-12p35 and IL-35

These mechanistic differences suggest that IL-12p35 and IL-35 may have complementary but distinct roles in immune regulation, with IL-12p35 functioning primarily as an antagonist of inflammatory cytokine signaling rather than through direct activation of suppressive pathways .

How can IL-12p35-induced regulatory cells be utilized in cell-based therapies?

IL-12p35's ability to induce regulatory cell populations offers promising avenues for cell-based immunotherapies:

• The capacity of IL-12p35 to convert conventional B cells into IL-10-producing and IL-35-producing regulatory B cells suggests its potential use for ex vivo expansion of regulatory cells for adoptive transfer therapies

• IL-12p35 treatment of encephalitogenic T cells rendered them partially anergic, suggesting potential applications in modulating pathogenic T cell responses

• In vivo treatment with IL-12p35 increased regulatory T and B cells in the CNS of EAE models, suggesting potential for targeted delivery to induce localized immunosuppression

The advantage of IL-12p35 over IL-35 for these applications lies in the technical simplicity of producing functional recombinant single-chain proteins compared to heterodimeric cytokines, making IL-12p35-based approaches potentially more feasible for clinical translation .

What key differences exist between human and mouse IL-12p35?

Understanding species differences is critical for translational research involving IL-12p35:

Table 4: Human vs. Mouse IL-12p35 Characteristics

These differences highlight the importance of careful interpretation when extrapolating findings from mouse models to human applications, particularly for therapeutic development .

How does the signal peptide processing of IL-12p35 impact cytokine production?

The unusual signal peptide processing of IL-12p35 represents a unique regulatory mechanism:

• Unlike typical cotranslational signal peptide removal, p35 preprotein enters the ER with its signal peptide intact

• The first cleavage occurs within the ER at the middle of the hydrophobic region of the signal peptide

• The preprotein becomes glycosylated upon ER entry, but glycosylation status does not affect this primary cleavage

• A second cleavage removes the remaining portion of the signal peptide, possibly involving a metalloprotease, concurrent with additional glycosylation and secretion

• Inhibition of glycosylation with tunicamycin prevents secretion of p35, while p40 secretion remains unaffected by glycosylation inhibition

This complex processing mechanism may provide additional control points for regulating IL-12 and IL-35 production, offering potential targets for therapeutic intervention in inflammatory and autoimmune conditions .

What are the critical unresolved questions about IL-12p35 function?

Several important questions remain to be addressed in IL-12p35 research:

• The receptor(s) through which monomeric or homodimeric IL-12p35 mediates its effects remains unidentified

• The molecular mechanisms by which IL-12p35 antagonizes STAT pathways induced by other cytokines require further elucidation

• The potential for IL-12p35 to generate peptide fragments with distinct biological activities during its unusual processing warrants investigation

• The differences in IL-12p35 function between various human cell types and disease states need comprehensive characterization

• The long-term effects of IL-12p35 treatment on immune surveillance and host defense require evaluation

What therapeutic potential does IL-12p35 hold beyond autoimmune diseases?

The immunoregulatory properties of IL-12p35 suggest potential applications beyond autoimmune diseases:

• Cancer immunotherapy: IL-35 promotes tumor growth and metastasis , suggesting IL-12p35 might modulate tumor immune environments through similar but potentially distinct mechanisms

• Transplantation: The ability to induce regulatory cell populations could be exploited to promote tolerance to allografts

• Infectious diseases: Balancing protective immunity while preventing immunopathology during chronic infections

• Inflammatory disorders: Conditions driven by dysregulated cytokine signaling might benefit from the STAT-antagonistic properties of IL-12p35