Anti-Mouse CD25 – Purified in vivo GOLD™ Functional Grade

Clone PC61 is primarily used in in vivo mouse studies to deplete CD25^+^ regulatory T cells (Tregs) and/or to block CD25 function, which directly impacts interleukin-2 (IL-2) signaling and immune homeostasis.

Key applications and mechanistic details:

• In vivo depletion of CD25^+^ Treg cells: PC61 is widely used to remove Tregs from mice by targeting CD25, the IL-2 receptor ? chain, on their surface. This depletion can cause aberrant immune activation, as Tregs are critical for maintaining immune tolerance.
• Blockade of IL-2 signaling: PC61 can also functionally block the binding of IL-2 to CD25 in vitro and in vivo, without actively depleting Tregs, depending on the antibody’s Fc variant. Studies have shown that immune homeostasis can be maintained when CD25 signaling is blocked rather than when Treg cells are depleted.
• Use of engineered variants: Modifications of the Fc region of PC61 (e.g., PC61-mIgG2a vs. PC61-mIgG1(N297Q)) allow researchers to control whether the antibody mediates cell depletion (via Fc effector functions) or simply blocks receptor signaling. Fc variants with strong effector functions induce depletion; those without (e.g., N297Q) primarily block signaling.

Additional experimental details:

• PC61 is a rat anti-mouse monoclonal antibody developed against an IL-2-dependent cytolytic mouse T-cell clone (B6.1).
• Applications include flow cytometry (FC), immunoprecipitation, in vivo Treg depletion, and functional inhibition of IL-2-dependent pathways.
• Storage, purity, and formulation details are standardized for experimental reproducibility.

In summary, PC61 is a tool for manipulating the Treg compartment or IL-2 pathway in mice, either by cellular depletion or functional blockade, depending on the experimental design and antibody variant used.

Proteins and Antibodies Commonly Used with PC61 in the Literature

PC61 is a widely used monoclonal antibody targeting mouse CD25 (interleukin-2 receptor alpha chain, IL-2R?). In the literature, PC61 is frequently employed alongside other proteins and antibodies, especially in studies investigating regulatory T cells (Tregs), immunoregulation, and immune cell phenotypes. Below are the most common co-used molecules and their biological roles.

Key Antibodies and Proteins

• Anti-CD4: Since PC61 targets CD25, which is highly expressed on CD4+ Tregs, anti-CD4 antibodies are routinely used in co-staining to identify and sort regulatory (CD4+CD25+Foxp3+) and conventional (CD4+CD25?) T cells in flow cytometry and cell depletion studies.
• Anti-Foxp3: Foxp3 is the master transcription factor for regulatory T cells. Staining with anti-Foxp3, together with anti-CD4 and anti-CD25 (PC61), is standard for identifying and characterizing mouse Treg populations.
• Other Anti-CD25 Clones (e.g., 3C7): While PC61 targets a specific epitope on mouse CD25, other clones such as 3C7 are also available and may be used in parallel for comparative studies or to confirm results, as PC61 and 3C7 recognize different epitopes.
• IL-2: The natural ligand for CD25, used in functional assays to study IL-2 signaling and T cell proliferation, often in combination with PC61 for blocking or modulation experiments.
• Anti-CD3 and Anti-CD28: Used in T cell activation and proliferation assays, often alongside PC61 to modulate CD25 expression or function.

Functional and Blocking Experiments

• IL-2 Binding Blocking: PC61 is frequently used to block IL-2 binding to its receptor, both in vitro and in vivo, to study the role of IL-2 signaling in immune responses.
• Depletion of CD25+CD4+ Tregs: In vivo, PC61 is used to deplete CD25+CD4+ Tregs, enabling researchers to study the impact of Tregs on immunity, autoimmunity, and inflammation.
• Growth Inhibition of IL-2-Dependent Cell Lines: PC61 can inhibit the growth of IL-2-dependent T cell lines, providing a tool to dissect IL-2 signaling pathways.

Additional Technical Notes

• Low and Ultra-Low Endotoxin Versions: For in vivo studies, low or ultra-low endotoxin versions of PC61 are recommended to minimize non-specific immune activation.
• Other Applications: PC61 is also used in immunoprecipitation, immunohistochemistry (IHC), and Western blotting, sometimes alongside antibodies against other cell surface markers for comprehensive phenotyping.

Summary Table

Conclusion

PC61 is most often paired with antibodies against CD4 and Foxp3 for Treg identification and functional studies, as well as with IL-2 for signaling assays. Other anti-CD25 clones, such as 3C7, are used for epitope comparison. For in vivo work, low endotoxin formulations are essential, and co-use with T cell activation markers (CD3/CD28) is common in mechanistic studies. These combinations are foundational in immunology research focused on regulatory T cells and IL-2 receptor biology.

The anti-mouse CD25 monoclonal antibody clone PC61 is a widely used tool in immunology research, primarily for studies targeting regulatory T cells (Tregs) by either blocking CD25 (IL-2 receptor ?-chain) function or depleting CD25? cells. Key findings from scientific literature citing clone PC61 include:

• Distinction between CD25 blockade and depletion:

  • Engineering Fc variants of PC61 allowed researchers to separate the effects of blocking CD25-mediated IL-2 signaling (without cell depletion) from active depletion of CD25? Treg cells by antibody effector function.
  • CD25 blockade with PC61 maintains immune homeostasis: When only IL-2 signaling is blocked (without depleting cells), immune regulation is preserved.
  • Depletion of CD25? Treg cells leads to immune activation: Antibody variants that induce depletion result in loss of regulatory T cells, driving aberrant immune activation and loss of tolerance.

• Mechanism of action:

  • PC61 blocks IL-2 binding to CD25, inhibiting high-affinity IL-2 receptor signaling.
  • Both effector-competent (able to deplete) and non-depleting (Fc-modified) PC61 variants bind CD25 and inhibit IL-2-induced STAT5 phosphorylation in Tregs equally well.

• PC61 as an experimental tool:

  • Used to modulate Treg cell populations in vivo, discerning the respective roles of cytokine signaling blockade versus physical depletion in immune regulation.
  • Findings emphasize the importance of antibody Fc engineering in determining whether a therapeutic antibody will merely block signaling or also cause target cell depletion.

• Relevance to clinical translation:

  • Insights from PC61 studies inform the design of anti-CD25 therapies in human medicine, particularly the distinction in outcome between blockade of signaling and depletion of regulatory T cells.

These findings highlight clone PC61's critical role in dissecting Treg biology and in shaping strategies for immunomodulatory antibody therapeutics targeting the IL-2/CD25 axis.

Dosing regimens of clone PC61 (anti-mouse CD25 monoclonal antibody) commonly involve intraperitoneal injections of 500??g per mouse every 7 days, but can vary depending on mouse strain and experimental conditions. For example, in studies involving wild-type and Fcer1g^?/? mice, PC61 was consistently dosed at this amount and frequency to maintain receptor saturation and effective CD25 blockade throughout both short (1-week) and long (4-week) experiments.

• Wild-type mice: 500??g/mouse, i.p., every 7 days; used in both single-week and 4-week dosing studies to saturate CD25.
• Fcer1g^?/? mice: Same regimen as wild-type, allowing comparative analysis of PC61’s effects across genotypes.
• Control groups: Typically receive PBS vehicle, not PC61.

In mouse models of experimental autoimmune encephalomyelitis (EAE), PC61 was given using this dosing schedule, with clinical monitoring and supportive care as needed based on disease severity.

Regimen variation considerations:

• Duration of treatment: Some studies use a single dose or doses over one week; others extend to four weeks, increasing group sizes for longer studies.
• Antibody variants: Both depleting (PC61-mIgG2a) and non-depleting (PC61-mIgG1^N297Q^) forms maintain the same basic dosing schedule; their pharmacodynamics differ.
• Knockout or transgenic mice (e.g., Fcer1g^?/?): No adjustments in dose or frequency were noted in the referenced experiments, but effects on immune cell numbers may differ between strains.

Published regimens typically validate dosing by demonstrating complete CD25 receptor saturation via flow cytometry at experiment termination. If different mouse models are used (immune-deficient, transgenic, autoimmune-prone), protocols may adjust dosing based on pilot pharmacokinetic results or desired immunological effects, though the 500??g/mouse/week regimen is a widely adopted starting point.

No evidence in the search results indicated major variations in dosing amount or interval for PC61 across mouse strains, but specific aims (Treg depletion/blockade, duration of immune modulation) and antibody isotype/variant may prompt investigators to modify the regimen. Always confirm dosing strategy with published studies specific to the disease model and mouse genetic background in use.