Anti-Mouse CD3ε [Clone 145-2C11] — Purified in vivo GOLD™ Functional Grade

The most common in vivo applications of clone 145-2C11 in mice are T cell depletion, T cell activation, and functional modulation in various immunological and disease models.

Key details:

• T cell depletion: 145-2C11 is widely used to deplete T cells in mouse models. This allows researchers to investigate the role of T cells in immunity, autoimmunity, transplantation, and infection by comparing outcomes with and without T cells.
• T cell activation: When administered in vivo, clone 145-2C11 can activate T cells by crosslinking the CD3 complex. This leads to T cell proliferation, cytokine release (such as IFN-γ, IL-2, TNF-α), and can model immune activation or cytokine release syndromes in mice.
• Functional modulation: The antibody is also used to modulate T cell–mediated immune responses for studying tolerance induction, autoimmune disease mechanisms, and T cell–driven pathology.
• Experimental disease models: Because of its potent effects on T cells, 145-2C11 is commonly used in mouse models of autoimmune diseases, graft-versus-host disease, cancer immunotherapy, and infectious disease to probe the role of T cell signaling and function.

Additional context:

• The effects of in vivo administration can vary with dose and format. Low doses may activate T cells, while higher doses may induce anergy, apoptosis, or depletion.
• The antibody is typically hamster IgG against mouse CD3ε and should be used with formulations of low endotoxin for in vivo work.
• For in vivo functional modulation, researchers sometimes use F(ab')₂ fragments of 145-2C11, which can mediate activation without Fc-dependent depletion.

In summary, the main in vivo uses of 145-2C11 in mice are for manipulating T cell numbers and function—either by depleting T cells or by stimulating T cell activation, to clarify their roles in health and disease.

Commonly used antibodies or proteins in combination with 145-2C11 (anti-CD3) in the literature include:

• Anti-CD4 antibodies: Often used to identify and analyze CD4+ T cell subsets by flow cytometry alongside 145-2C11.
• Anti-CD8 antibodies: Similarly applied for immunophenotyping of CD8+ T cell subsets, typically in conjunction with 145-2C11.
• Concanavalin A (Con A): A plant lectin frequently used as a non-specific T cell stimulant in functional assays together with 145-2C11 to assess T cell proliferative responses.
• Steroid hormones (glucocorticoids, e.g., methylprednisolone): Utilized in protocols that involve 145-2C11 to modulate cytokine release syndrome in mice.

Additional markers and reagents that may be used with 145-2C11 based on experimental design:

• Anti-mouse CD3ε F(ab')₂ fragments: Used for experiments requiring the CD3 signal without engaging Fc receptors or activating accessory cell functions.
• Anti-CD25 or anti-Foxp3 antibodies: To further delineate regulatory T cell populations in analyses containing 145-2C11, though these are less frequently reported in the provided results.
• Fc receptor-bearing accessory cells: Included in some in vitro settings to facilitate cross-linking and optimal stimulation when using the full-length antibody.

In summary, anti-CD4 and anti-CD8 antibodies are the principal molecules co-used with 145-2C11 for immunophenotyping T cell subsets. Under certain conditions, functional assays may also include Con A, and protocols aimed at modulating immune responses may involve glucocorticoids. Use of F(ab')₂ fragments is noted when Fc region effects are undesirable.

Clone 145-2C11 is a widely studied hamster monoclonal antibody that targets the mouse CD3ε chain of the T cell receptor (TCR) complex. Its key findings and uses in the scientific literature include:

• Potent T Cell Activation and Modulation:

  • 145-2C11 binding to CD3ε induces T cell activation, cellular proliferation, and can also lead to apoptosis depending on the experimental context.
  • This activation is exploited in vitro and in vivo to study T cell signaling, activation thresholds, and immune responses.

• Essential Research Tool for Mouse Immunology:

  • 145-2C11 is extensively used as a T cell marker in flow cytometry and immunohistochemistry, allowing reliable identification and quantitation of mouse T cells due to its specific recognition of the CD3ε subunit.
  • It is standard in protocols for both basic characterization and advanced functional assays involving mouse T cells.

• Therapeutic Proof-of-Concept in Autoimmunity:

  • Pioneering studies showed that intact 145-2C11 can cure new-onset autoimmune diabetes in NOD mice, with cure rates of 64-80% after short-term treatment immediately after disease onset.
  • However, its efficacy is coupled with significant cytokine release and mitogenic activity due to Fc receptor (FcR) engagement, limiting its clinical translation in unmodified form.

• Mechanistic Insights and Modified Variants:

  • Both 145-2C11 and F(ab')2 fragments can rapidly downregulate surface TCR/CD3 on T cells, which can be used for studies of TCR signaling and tolerance induction.
  • Engineered derivatives like "2C11-Novi" lacking FcγR binding retain TCR downregulating and immunomodulatory activity, but evade systemic cytokine release, highlighting the role of FcR interaction in the antibody's mitogenic effects.
  • Recovery kinetics of TCR expression differ between intact and Fc-deficient forms, offering further insight into mechanisms of action.

• Context and Limitations:

  • The antibody is strictly specific for mouse CD3ε and thus not applicable to human T cell studies.
  • Its strong activation properties require careful dosing and are sometimes leveraged to induce apoptosis or immune modulation in experimental models.

In summary, clone 145-2C11 is a cornerstone reagent in murine T cell immunology for both phenotyping and functional studies, and its use has delivered crucial insights into immune activation, tolerance, and therapeutic strategies for autoimmunity, with an important body of literature also focusing on variants designed to separate efficacy from adverse systemic cytokine release.

Dosing regimens of clone 145-2C11 in mouse models vary widely based on the experimental goals, mouse strain, disease model, and whether the whole antibody or fragment is used. Typical doses range from as low as 1–5 μg up to 400 μg per mouse, delivered as either a single or multiple injections, most often intraperitoneally.

Essential context and supporting details:

• Mouse Strain Differences:

  • In BALB/c mice, regimens have used single doses (e.g., 20–400 μg), or multiple doses such as five injections of 50 μg every 24 hours (total: 250 μg), or four doses of 25 μg every 72 hours (total: 100 μg).
  • In NOD mice (diabetes model), regimens included five doses of 50 μg every 24 hours, four doses of 25 μg every 72 hours, and much lower doses (as little as 1 μg).
  • Outcomes such as TCR modulation and diabetes remission were tracked to assess efficacy.

• Purpose and Endpoint:

  • For immunosuppression, doses at the upper end (250–400 μg) are used for strong T cell depletion or modulation.
  • For transient immune modulation, lower or spaced doses (down to 1–5 μg per injection) are employed, especially in studies needing sustained but partial effects.

• Comparison—Single vs. Multiple Doses:

  • Single high doses (e.g., 400 μg) deplete up to 40% of CD4+ T cells rapidly.
  • Multiple lower doses (e.g., 25–50 μg daily for 3–5 days) allow for controlled depletion and recovery of T cells and Tregs, suiting chronic models or when sustained modulation is needed.

• Formulation—Whole Antibody vs. Fragments:

  • Whole antibody (145-2C11 IgG) induces potent activation and cytokine release.
  • F(ab’)_2 fragments are used for studies aiming to reduce Fc-mediated effects and cytokine storm; these fragments can be similarly dosed (often 25–50 μg per injection), but may require consecutive dosing for comparable immune modulation.

• Tumor and Autoimmune Models:

  • Dosing is further modified by disease context and immune cell targets. For example, autoimmune models (e.g., diabetes in NOD mice) may need repeated dosing at lower levels to avoid adverse events while achieving therapeutic effects.

Additional relevant information:

• Administration Route: Intraperitoneal injection is standard for most regimens.
• Recovery Kinetics: Some regimens favor rapid recovery of T cell populations, depending on study needs (e.g., earlier recovery with trivalent non-Fc constructs as compared to bivalent whole antibodies).

Summary Table—Representative Regimens in Mouse Models:

Dosing should be tailored to model, disease context, and desired immune modulation. Researchers frequently adjust regimens based on pilot data, toxicity, and experimental endpoints.