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

The PC61 clone is a rat anti-mouse CD25 monoclonal antibody widely used in in vivo mouse studies to deplete or functionally block CD25(^+) regulatory T cells (Tregs), or to block the IL-2 signaling pathway.

Key Uses in In Vivo Mouse Studies:

• Depletion of CD25(^+) Treg Cells:
  PC61 is frequently administered to mice to deplete CD25(^+) Treg cells through its Fc-mediated effector functions, thereby enabling researchers to investigate the role of Tregs in immune homeostasis, autoimmunity, infection, and tumor immunity. The capacity to deplete isotype and Fc variant dependent; for example, some engineered versions of PC61 more effectively induce antibody-dependent cell-mediated cytotoxicity (ADCC), leading to robust Treg depletion.

• Blockade of IL-2 Signaling Without Depletion:
  In some contexts, PC61 can be used in forms (e.g., specific Fc variants) that block IL-2 binding to CD25 without depleting the Treg population, allowing a functional inhibition of Treg activity rather than their physical removal.

• Functional Assays:
  In vivo, PC61 has been used to study immune activation resulting from either physical depletion or simple blockade of Tregs and to distinguish the immune consequences of these two mechanisms. For example, sustained depletion (e.g., with more cytotoxic PC61 Fc variants) leads to T cell activation and immune dysregulation, whereas blockade alone maintains some immune homeostasis.

• Dosing and Applications:
  PC61 is available in several recombinant and purified formats and can be administered in vivo via intraperitoneal injection at investigator-determined doses and schedules. Detailed application notes and titration recommendations can vary by supplier and study goals.

Summary Table:

Important Considerations:

• The exact outcome—whether depletion, blockade, or both—depends on the antibody isotype, Fc modifications, and dose.
• There is ongoing controversy about the exact in vivo depletion efficiency and mechanism of PC61, with results sometimes varying between studies or mouse strains.
• Controls such as isotype antibodies and different Fc variants are recommended to distinguish effects due to depletion vs. signaling blockade.

PC61 clone is a versatile tool but experimental outcomes are influenced by the specific format and experimental setup chosen.

Commonly used antibodies or proteins alongside PC61 (anti-mouse CD25) in the literature include other markers for immune cell characterization and depletion experiments. Prominent examples are:

• Anti-CD4: Used to identify and sort CD4(^+) T cells, including regulatory T cells (Tregs) that express CD25, the target of PC61.
• Anti-CD8: Labels cytotoxic T cells, often included for comprehensive T cell profiling in flow cytometry.
• Anti-Foxp3: An intracellular marker for Tregs, typically used in combination with surface staining for CD4 and CD25 to confirm regulatory phenotype.
• Anti-CD3: Pan-T cell marker, frequently used to identify the total T cell population.
• Anti-CD45: A general leukocyte marker, sometimes included for gating strategies in immunophenotyping panels.

In functional or depletion studies using PC61, antibodies such as 3C7 (another anti-CD25 clone, but with different epitope recognition) are referenced for comparative or validation purposes.

For in vivo experiments aiming to deplete regulatory T cells, PC61 is routinely used with:

• Isotype controls: To validate specificity and rule out nonspecific effects.
• Anti-IL-2 or IL-2 immune complexes: Occasionally, studies use blocking or neutralizing IL-2 in tandem with PC61 for mechanistic investigations.

These combinations optimize identification, depletion, or characterization of T cell subsets, especially Tregs, in various research contexts (e.g., immunophenotyping, depletion assays, functional studies).

Alternative clones or reagents mentioned for similar applications:

• 3C7: Recognizes a different CD25 epitope than PC61 and is sometimes preferred for sensitive assays or comparative studies.
• Ultra-LEAF™ purified PC61: For endotoxin-sensitive applications, often used in in vivo Treg depletion experiments.

PC61 is rarely used alone; typical panels include several antibodies for multi-parameter flow cytometry or depletion studies to ensure comprehensive analysis of immune cell subsets.

The key findings from scientific literature citing clone PC61 center on its effects as an anti-CD25 monoclonal antibody used to study and manipulate regulatory T cells (Tregs), especially in mouse models. The most important insights are:

• PC61 blocks CD25 and inhibits IL-2 signaling: Clone PC61 binds to mouse CD25 (IL-2 receptor alpha subunit), effectively blocking IL-2 from binding and thereby inhibiting high-affinity IL-2 receptor signaling, as measured by reduced STAT5 phosphorylation in Treg cells.

• Distinction between CD25 blockade and Treg cell depletion: Engineering different Fc variants of PC61 showed that the immune effects depend on whether PC61 only blocks CD25 signaling or also depletes CD25(^+) Treg cells via Fc-mediated effector function. When CD25(^+) Tregs are simply blocked (not depleted), immune homeostasis is generally maintained. In contrast, active depletion of CD25(^+) Treg cells leads to aberrant immune activation, indicating that Treg depletion disrupts immune regulation more than blockade alone.

• Implications for therapeutic design: These findings inform the design of therapeutic antibodies targeting CD25, suggesting that selective blockade—without depletion—is preferable for maintaining immune balance, while depletion may trigger autoimmune-like pathology.

• Methodological advances: The literature details cloning, engineering, and production of PC61 variants, confirming variable region identity and antibody functionality in blocking IL-2 signaling, which establishes the experimental rationale for using PC61 in immunological studies.

In summary, clone PC61 is a crucial tool for dissecting the roles of CD25 and Treg cells in immune regulation, with scientific findings emphasizing the difference between mere signaling blockade and physical depletion of Tregs, as well as implications for antibody-based immunotherapies.

Dosing regimens of clone PC61, an anti-mouse CD25 monoclonal antibody, typically involve intraperitoneal injection of 500??g per mouse every 7 days, but can vary depending on the mouse strain, immunological context, and experimental objectives.

• In wild-type and Fcer1g^?/?^ mice, PC61-mIgG2a and PC61-mIgG1(N297Q) variants were administered at 500??g/mouse, once weekly via intraperitoneal injection.
• For short-term (1-week) studies, this dosing schedule is commonly used in groups of five mice per treatment. Longer studies (4 weeks) may increase the group size, but the dosing frequency and amount remain unchanged.
• This regimen is designed to maintain complete receptor saturation, as confirmed by flow cytometry of splenocytes at the end of experiments.

Additional Sources:

• Guidance from comprehensive antibody dosing guides generally supports doses in the 100–500??g/mouse range for mouse monoclonal antibodies targeting lymphocyte populations, administered intraperitoneally every 3–7 days, contingent on the antibody’s half-life and depletion efficacy.
• Functional efficacy can vary by mouse model: For example, PC61 in the A20 lymphoma model failed to inhibit tumor growth when administered after tumors became palpable, possibly due to the antibody’s isotype and mouse Fc receptor expression, indicating that dosing regimens optimized for Treg depletion may not yield anti-tumor effects in all contexts.

Key Factors Influencing Regimen Variation:

• Mouse strain or genetic background: Strains with altered Fc receptor expression (e.g., Fcer1g knockout) may require adjustment in dosing or choice of PC61 variant.
• Duration of study: While weekly dosing is standard, acute studies may use a single injection, whereas chronic studies retain regular dosing to sustain depletion.
• Experimental endpoint: For Treg depletion, maintaining receptor saturation is critical; for tumor or autoimmune models, additional dosing (e.g., peri-tumor challenge) or altered schedules may be needed depending on disease kinetics.
• Antibody modifications: Chimeric or Fc-engineered PC61 variants (e.g., mIgG2a, mIgG1[N297Q]) can alter pharmacokinetics or effector function, necessitating regimen adjustment.

In summary, the most common regimen for clone PC61 is 500??g/mouse weekly by intraperitoneal injection, but doses and schedules can be tailored to the mouse model, antibody variant, and study aims, with frequent confirmation of CD25 receptor coverage and immune cell depletion via flow cytometry.