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

In Vivo Applications of Clone PC61 in Mice

Clone PC61 (a monoclonal antibody targeting mouse CD25, the alpha chain of the interleukin-2 receptor) is primarily utilized in vivo for specific immunomodulatory interventions in mouse models.

Depletion of CD25+ Regulatory T Cells (Tregs)
The most common application is the selective depletion of CD25+CD4+ Foxp3+ regulatory T cells (Tregs) in vivo. Administering PC61 via intraperitoneal injection has been shown to dramatically reduce (95–98%) CD25+ CD4+ T cells in organs such as the spleen, lungs, and mediastinal lymph nodes (mLNs). This is a well-established method to study the role of Tregs in immune responses, tolerance, autoimmunity, and infection models.

Blockade of IL-2 Signaling
Beyond depletion, PC61 can block interleukin-2 (IL-2) binding to its receptor, directly interfering with IL-2-mediated signaling pathways that are critical for T cell proliferation and immune homeostasis. This application is valuable for investigating the contributions of IL-2 signaling to immune cell function and survival in vivo.

Modulation of Immune Responses in Infection Models
PC61 has been used to examine the impact of Treg depletion on antiviral immune responses. For example, pre-treatment with PC61 prior to viral challenge (e.g., LCMV infection) alters the dynamics of virus-specific CD8+ T cell responses, though the effects can differ depending on the infection history of the mice (naive vs. pre-immune). In some cases, PC61 treatment has been associated with reduced lung pathology without affecting viral titers, highlighting context-dependent roles of Tregs in immunopathology.

Experimental Protocols

• Dosage and Timing: A typical protocol involves a single intraperitoneal injection of 100 μg PC61, administered 3 days prior to the experimental intervention (e.g., infection).
• Controls: Isotype control antibodies or PBS are used to ensure specificity of the observed effects.
• Validation: Efficacy is confirmed by flow cytometry, showing near-complete depletion of CD25+CD4+ T cells in relevant tissues.

Summary Table

Key Points

• PC61 is a tool for in vivo depletion and functional blockade of CD25+ Tregs in mice.
• It is widely used to dissect the roles of Tregs and IL-2 signaling in immune homeostasis and disease models.
• Effects are context-dependent, especially in infection models, underscoring the need for appropriate controls.

Clone PC61 remains a cornerstone reagent for in vivo immunological research in mice, enabling precise manipulation of Treg populations and IL-2 signaling to uncover their roles in health and disease.

In the literature, the PC61 antibody is commonly used in conjunction with other antibodies and proteins for various applications, particularly in immunological studies. Here are some of the commonly used antibodies or proteins:

1. CD4 and Foxp3 Antibodies: These are used alongside PC61 for the identification and functional studies of regulatory T cells (Tregs).

2. IL-2 (Interleukin-2): PC61 is paired with IL-2 for signaling assays due to its role in blocking IL-2 binding to the CD25 receptor.

3. Other Anti-CD25 Clones: Clones like 3C7 and 7D4 are used for epitope comparison and studying different aspects of CD25 function.

Additionally, PC61 is often used in flow cytometric analysis, which may involve other antibodies for multi-color staining and identification of different cell populations.

These combinations enhance the understanding of immune cell dynamics, particularly in mouse models where PC61 is extensively used due to its ability to block IL-2 signaling, which is crucial for Treg depletion in various research contexts.

Clone PC61 is a rat monoclonal antibody widely used in mouse models to target CD25 (the interleukin-2 receptor alpha chain) and regulate regulatory T cell (Treg) biology, but its precise mechanism and impact have been clarified in recent literature.

Key findings from PC61 clone citations:

• Dual Mechanism: Blockade and Depletion

  • PC61 can effect both blockade of IL-2 signaling through CD25 and depletion of CD25^+^ Treg cells, but these are distinct outcomes determined by isotype and Fc engineering of the antibody.
  • Fc-engineered variants of PC61 demonstrated that immune homeostasis can be maintained when only IL-2 signaling is blocked, but active depletion of CD25^+^ Treg cells disrupts homeostasis and leads to immune activation.

• Mechanism of Depletion

  • PC61-mediated depletion relies largely on Fcγ receptor III (FcγRIII)-positive phagocytes, specifically macrophages, not NK cells.
  • Functional blockade of Fcγ receptors or depletion of phagocytes abolishes the Treg depletion effect of PC61.

• Effectiveness and Limitations

  • In vivo studies often show incomplete depletion (typically 30–70%) of CD25^+^ Treg cells, and residual Treg often display lower or absent CD25 expression.
  • The rat IgG1 isotype of PC61 has weak effector function in mice; thus, depletion is sometimes inefficient or inconsistent, which has led to misinterpretations in earlier studies regarding its true impact.
  • Fc-optimized (e.g., murine IgG2a) versions show stronger depletion and improved anti-tumor efficacy—but often require combination therapy (such as with anti-PD1) for significant effects in cancer models.

• Functional Blockade Without Depletion

  • Non-depleting variants of PC61 (e.g., mouse IgG1 or with N297Q mutation) block IL-2-mediated STAT5 phosphorylation but do not lead to Treg cell elimination.
  • Even with CD25 blockade, some residual CD25 function may persist, helping maintain Treg-mediated immune homeostasis.

• Implications

  • There's now a clear distinction between Treg depletion (affecting both numbers and function and potentially causing immune activation) and pure blockade (mainly affecting IL-2 signaling without massive Treg loss).
  • These findings highlight the importance of antibody isotype and Fc engineering in the design of anti-CD25 therapeutics and the interpretation of Treg depletion experiments.

Summary Table: Functional Outcomes of PC61 Use

Conclusion:
The literature consistently shows that PC61's impact varies with its Fc characteristics: it can act as a depleting or blocking antibody, with depletion requiring FcγRIII^+^ phagocytes. This distinction is crucial for experimental interpretation and for the rational design of therapies targeting regulatory T cells.

Dosing regimens of clone PC61 (anti-mouse CD25) in mouse models most commonly use 500 µg per mouse, administered by intraperitoneal injection at weekly intervals, but specific dosing and schedules are often adjusted based on mouse strain, experimental goal, and length of study.

• In standard immunology and autoimmunity models (e.g., Treg cell depletion, IL-2 biology, experimental autoimmune encephalomyelitis), PC61 is typically dosed at 500 µg per mouse intraperitoneally, once weekly. This schedule aims to maintain consistent receptor saturation as confirmed by flow cytometry.
• The regimen is consistent across both wild-type and genetically modified mice, such as Fcer1 g^−/−^ strains, with this dose and interval providing effective CD25 blockade.
• For longer experiments (e.g., 4-week studies), dosing remains at 500 µg per week but may use more animals per group to increase statistical power.
• The same dosing strategy is reported in experimental autoimmune encephalomyelitis models, with ongoing clinical monitoring according to the severity of disease progression.
• Manufacturers and antibody suppliers also cite 500 µg/mouse/week as the most common regimen for in vivo depletion, but acknowledge that variability exists and regimens can be tailored to mouse model and study design. Alternate schedules, higher frequency, or dose adjustments may occur in models with different pharmacokinetics or when aiming for partial vs. full Treg depletion.

Variability between models:

• Some investigators may use higher or lower doses, or more frequent dosing in certain tumor models, particularly where faster or deeper Treg depletion is required, or where the pharmacokinetics of the mouse strain support altered timing.
• Antibody isotype variant (e.g., chimeric murine IgG2a vs. rat IgG1) can affect depletion efficacy, especially in tumor models; PC61-mIgG2a variants may be required for improved Treg clearance in certain contexts.

Summary Table: PC61 Dosing in Common Mouse Models

Key factors influencing regimen selection:

• Mouse strain (C57BL/6, BALB/c, KO models)
• Experimental duration
• Desired degree of Treg depletion
• Fc isotype optimization in tumor models for Treg clearance efficacy

The default and most cited regimen remains 500 µg/mouse weekly i.p., but researchers optimize schedules based on their specific experimental models and endpoints.