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

Clone PC61 is most commonly used in vivo in mice for depleting CD25⁺ regulatory T cells (Tregs) and/or blocking CD25 (IL-2Rα) function, thereby affecting interleukin-2 (IL-2) signaling and immune regulation.

Key in vivo applications include:

• Depletion of CD25⁺ Treg cells: PC61 is widely employed to reduce or eliminate immunosuppressive Treg populations in mouse models, which is a standard technique for investigating the role of Tregs in various immune responses, autoimmunity, tumor immunity, and transplantation.
• Functional blockade of CD25: PC61 can block IL-2 binding to its receptor, interfering with IL-2–dependent T cell proliferation and function. This allows for the study of how IL-2 signaling influences immune homeostasis and effector T cell responses.
• Modulation of immune responses: By depleting or inhibiting CD25⁺ cells, PC61 is used to assess Treg contributions in models of infection, cancer, allergy, and autoimmunity.

Additional, but less common, in vivo applications:

• Growth inhibition of IL-2–dependent T cell lines: In some studies, PC61 is used to limit proliferation of cells relying on IL-2 via CD25.
• Augmentation of immune-based therapies: PC61 can be used to improve the efficacy of vaccines or immunotherapies by removing CD25⁺ Tregs that otherwise dampen immune responses.

In summary, the main in vivo uses are Treg depletion and CD25 functional blockade to probe the roles of IL-2 signaling and regulatory cells in mouse immune models.

PC61, a rat anti-mouse CD25 monoclonal antibody, is frequently used in combination with several other antibodies and proteins in immunological research and functional studies.

Primary Antibody Combinations

PC61 is most often paired with antibodies against CD4 and Foxp3 for regulatory T cell (Treg) identification and functional studies. This combination is particularly important because CD25, CD4, and Foxp3 together serve as key markers for identifying and characterizing regulatory T cell populations in mouse models.

Signaling and Functional Assays

In studies examining IL-2 signaling pathways, PC61 is commonly used alongside IL-2 itself. This pairing is logical given that PC61 targets CD25, which is the alpha chain of the IL-2 receptor, and many studies investigate how blocking this interaction affects cellular responses and immune function.

Epitope Mapping Studies

For epitope comparison and validation purposes, PC61 is often used in conjunction with other anti-CD25 clones, such as 3C7. These comparative studies help researchers understand the different binding sites and functional consequences of various anti-CD25 antibodies.

Related Receptor Components

Since CD25 functions as part of the IL-2 receptor complex, research involving PC61 frequently examines other components of this receptor system, including the IL-2 receptor beta chain (IL2RB) and gamma chain (IL2RG). Together, these chains form the high-affinity IL-2 receptor, making them relevant partners in studies using PC61.

Clone PC61 is a rat anti-mouse monoclonal antibody widely used to target CD25, the high-affinity receptor for IL-2, in studies of regulatory T cell (Treg) biology in mice. Key scientific findings from literature citing PC61 center around its dual actions—functional blockade versus depletion—and their distinct immunological outcomes.

• Distinction Between CD25 Blockade and Depletion:

  • PC61 can either block IL-2 signaling (by binding to CD25) without depleting Tregs, or actively deplete CD25+ Tregs depending on its Fc variant. Fc-engineered variants that lack effector function (e.g., N297Q mutant) block signaling but do not deplete cells.
  • CD25 blockade (without Treg depletion) impairs IL-2 signaling but generally maintains immune homeostasis; immune dysregulation arises when Tregs are actively depleted.

• In Vivo Functional Impact:

  • Non-depleting PC61 variants (e.g., N297Q) reduce Treg sensitivity to IL-2 by about 100-fold but residual function is sufficient to maintain immune homeostasis. Even with partial blockade, Tregs still outcompete other lymphocytes for IL-2 access.
  • Depleting variants provoke immune activation due to loss of Treg-mediated suppression, emphasizing the need for careful antibody engineering when targeting CD25 therapeutically.

• Cancer and Immunotherapy Context:

  • Standard PC61 (rat IgG1) has limited efficacy in tumor models due to insufficient Treg depletion—high FcRIIb expression and poor Fc interactions restrict its activity against tumor-infiltrating Tregs.
  • Fc-optimized variants (e.g., murine IgG2a) exhibit more robust depletion especially in combination with other therapies (e.g., anti-PD-1).

• Methodological Considerations:

  • Past studies using PC61 often conflated functional inactivation (blocking IL-2 survival signals) with true depletion. It’s now established that the extent and mechanism of Treg reduction depend on PC61’s Fc characteristics.

Summary Table: Functional Outcomes of PC61 Variants

• PC61 has become the gold standard for defining Treg function in vivo, but results depend on antibody variant and experimental context.

These findings advise great caution in interpreting results from PC61 studies and inform the design of CD25-targeted therapies, as blocking alone may preserve enough Treg function for immune regulation, but depletion disrupts it and risks autoimmunity.

Dosing regimens of clone PC61, a rat anti-mouse CD25 monoclonal antibody, show considerable variation across different mouse models and experimental contexts, though a standard protocol has emerged as the most common approach.

Standard Dosing Protocol

The most widely used regimen for PC61 involves 500 μg per mouse administered weekly via intraperitoneal injection. This dose and frequency were determined through pharmacokinetic studies designed to maintain complete receptor saturation throughout the treatment period. The intraperitoneal route is preferred because it allows for minimal handling of mice while ensuring consistent delivery.

Variations in Duration and Frequency

The duration of PC61 treatment varies significantly depending on the experimental model. Short-term studies may involve 1-week dosing regimens, while chronic studies can extend to 4-week protocols with the same weekly 500 μg dose. In some experimental contexts, the dosing schedule is adjusted to maintain specific biological effects rather than following a rigid weekly pattern.

Model-Specific Considerations

Tumor Models: In cancer immunotherapy studies, PC61 administration timing is particularly critical. Anti-mouse CD25 monoclonal antibodies are most effective when injected before tumor inoculation or during early tumor stages. This timing constraint reflects the antibody's mechanism in depleting regulatory T cells (Tregs) to enhance anti-tumor immunity.

Autoimmune Disease Models: For experimental autoimmune encephalomyelitis (EAE) studies, PC61 dosing follows the standard 500 μg weekly protocol, but mice require daily monitoring for clinical disease scores and may need supportive care adjustments based on disease severity.

Fc Variant Considerations

The choice of PC61 Fc variant significantly impacts dosing outcomes. PC61-mIgG2a variants demonstrate depleting activity in wild-type mice, causing significant decreases in both absolute numbers and percentages of Treg cells. In contrast, PC61-mIgG1(N297Q) variants, which lack Fc receptor binding capability, show reduced depletion effects, with only percentage decreases in Treg cells but not absolute numbers. This distinction is crucial when designing experiments where IL-2 signaling blockade without Treg depletion is desired.

Flexibility in Dosing

While 500 μg per mouse weekly represents the standard approach, researchers can tailor doses and schedules to their specific experimental needs. The key consideration is maintaining sufficient receptor saturation, which should be confirmed through flow cytometry analysis of splenocytes at experiment termination.