Anti-Mouse CD134 (Clone OX-86) – Purified in vivo GOLD™ Functional Grade

Common In Vivo Applications of Clone OX-86 in Mice

Clone OX-86 is a monoclonal, rat IgG1 antibody that specifically targets mouse OX40 (also known as CD134), a member of the tumor necrosis factor (TNF) receptor superfamily expressed primarily on activated CD4+ and CD8+ T cells. OX-40 is not found on resting naïve T cells and is only minimally expressed on most resting memory T cells, but is also present on activated regulatory T cells (Tregs), NK cells, NKT cells, and neutrophils.

Immunomodulation and T Cell Activation

• Enhancement of T Cell Responses: OX-86 is widely used as an agonist antibody to stimulate the OX40 signaling pathway, leading to enhanced clonal expansion and prolonged proliferation of antigen-specific effector T cells. This stimulation can prevent the induction of T cell tolerance and boost immune responses against pathogens or tumors.
• Modulation of Immune Regulation: By engaging OX40, OX-86 can modulate immune regulation, promoting both effector and regulatory T cell responses, although its effects on Tregs are reported to be weaker compared to conventional T cells.
• Delay of Tumor Growth: In vivo treatment with OX-86 has been demonstrated to delay tumor growth, likely through the enhanced generation and activity of tumor-specific T cells.

Experimental Models

• Cancer Immunotherapy Research: OX-86 is frequently employed in mouse models of cancer to study the role of costimulatory signals in anti-tumor immunity and to evaluate potential immunotherapeutic strategies.
• Infectious Disease Models: The antibody is used to investigate mechanisms of protective immunity and memory formation in response to infections, leveraging its ability to amplify antigen-specific T cell responses.
• Autoimmunity and Tolerance Studies: Researchers utilize OX-86 to probe the balance between immune activation and tolerance, given OX40’s role in both promoting effector responses and, under certain conditions, influencing regulatory pathways.

Mechanistic Insights

• Costimulation of Naïve T Cells: OX-86 provides a costimulatory signal that, in conjunction with T cell receptor (TCR) engagement, leads to enhanced proliferation and cytokine production by T cells.
• Impact on Memory and Effector Populations: Studies have shown that OX40 signaling via OX-86 can affect the generation and maintenance of memory T cell populations, with notable effects observed in the context of MHC class II knockout mice.
• Isotype-Dependent Effects: The rat IgG1 isotype of OX-86 (as opposed to mouse IgG2a variants) is thought to mediate its effects primarily through agonism rather than Fc receptor-mediated depletion, due to its preferential binding to FcγRIIb and FcγRIII.

Technical Notes

• Administration: OX-86 is typically administered intraperitoneally or intravenously in experimental settings.
• Functional Grade: The antibody is available in specialized in vivo grades (e.g., PLATINUM™, GOLD™) to ensure low endotoxin levels and high purity for animal studies.

Summary Table

In summary, clone OX-86 is a versatile tool in mouse immunology, primarily used to stimulate OX40-dependent T cell activation, enhance anti-tumor and anti-pathogen immunity, and explore the mechanisms of immune regulation in vivo.

OX-86 (clone OX-86) is an agonistic antibody targeting mouse CD134 (OX40), widely used in immunological research, particularly for studying T cell activation and immune checkpoint modulation. In the literature, OX-86 is commonly combined with several other antibodies and proteins for both functional and mechanistic studies.

Common antibodies and proteins used with OX-86:

• Cytokine-detecting antibodies:
  Frequently used in assays measuring T cell effector functions after OX-86 stimulation, including:

  • Anti-IFN-γ
  • Anti-IL-2
  • Anti-IL-4
  • Anti-IL-17A
    These are typically employed in ELISA, intracellular cytokine staining, or similar assays to assess changes in cytokine production following OX-86-mediated activation.

• T cell marker antibodies:
  For flow cytometry or immunophenotyping, these are often used alongside OX-86 to delineate T cell subsets:

  • Anti-CD4
  • Anti-CD8
  • Anti-CD25
    This allows analysis of OX40 expression and function specifically within distinct T cell populations.

• OX40 ligand fusion proteins (OX40L, CD252):
  OX40L fusion proteins have been used to study interactions or compare OX-86 effects with direct OX40 ligand engagement.

• Checkpoint inhibitors and other immunomodulatory agents:
  In cancer and checkpoint studies, OX-86 is often used in combination with:

  • Anti-PD-1
  • Anti-CTLA-4
  • Other agents that modulate immune response pathways, as OX-86 can synergize with these in tumor models.

• Other immune cell markers (in some studies):

  • Anti-B220 (B cells)
  • Anti-NK1.1 (NK cells)
  • Anti-CD19
    These may be used for comprehensive immune profiling, particularly in studies of immune modulation or tumor microenvironment.

Typical applications:

• Flow cytometry: OX-86 is often paired with lineage and activation marker antibodies (e.g., CD4, CD25) for phenotyping activated T cells.
• Functional assays: Accompanied by cytokine detection antibodies (e.g., IFN-γ, IL-2) for measuring effector responses after antibody stimulation.
• In vivo synergy studies: Combined with other checkpoint inhibitors, chemotherapy, or immunotherapies to evaluate effects on tumor growth, memory T cell pool, or autoimmunity.

In summary, anti-cytokine antibodies (IFN-γ, IL-2, IL-4, IL-17A) and T cell markers (CD4, CD8, CD25) are most frequently used with OX-86 for functional and mechanistic studies, while checkpoint inhibitors and OX40 ligand proteins are common co-treatments in immunomodulation and oncology research.

Clone OX-86 is a monoclonal antibody targeting murine CD134 (OX-40, TNFRSF4) that has played an instrumental role in elucidating the functions of this critical immune checkpoint molecule. Research utilizing this clone has revealed several important insights into T cell biology and anti-tumor immunity.

Role in T Cell Activation and Memory Formation

OX-86 research has demonstrated that CD134/OX-40 is essential for optimal T cell responses. When CD134 is bound by its corresponding ligand (OX-40L), an optimal T cell response is generated that plays a significant role in determining the amount of memory T cells remaining after the immune response. Studies using OX-40 knockout mice showed that these animals generate fewer primary effector CD4 T cells after immunization, highlighting the importance of this pathway in T cell clonal expansion.

The antibody has been used to show that OX-40 provides a costimulatory signal to antigen-reacting naive T cells to prolong proliferation and augment the production of several cytokines. Treatment with OX-86 as an agonist antibody has been shown to strongly enhance the generation of antigen-specific effector T cells and prevent the induction of T cell tolerance.

Anti-Tumor Immunity

Clone OX-86 has been particularly valuable in demonstrating CD134's role in carcinogenesis and anti-tumor immunity. The antibody has been used as an agonistic agent that delays tumor growth in vivo. Research using OX-86 showed that treatment with activating in vivo antibodies against CD134 enhanced tumor responses, suggesting that CD134 functions as an important tumor suppressor, and its absence disrupts the immune response to tumors.

Expression Patterns and Cellular Distribution

Studies with OX-86 have revealed that CD134 is expressed on activated CD4 and CD8 T cells, but not on resting naive T cells or most resting memory T cells. The research has also expanded our understanding beyond conventional T cells, showing that OX-40 is expressed on activated regulatory T cells, B cells, NKT cells, NK cells, and neutrophils.

Functional Mechanisms

Research has shown that depletion of CD134 in murine models leads to reduced CD4+ and CD8+ T cell populations. The OX-86 antibody significantly increased the production of IL-2 and IFN-γ from immune cells, demonstrating its agonistic properties. Studies have also examined how different isotypes of the antibody interact with Fcγ receptors, with the rat IgG1 version interacting with only FcγRIIb and FcγRIII, giving it distinct functional properties compared to other isotypes.

Dosing regimens of clone OX-86 (anti-mouse OX40 monoclonal antibody) differ significantly depending on the mouse model, disease context, and experimental aim, commonly ranging from 10 µg to 250 µg per injection, with variable frequencies and durations.

Key findings by mouse model and experimental design:

• Basic Range: Typical doses are 10 µg to 250 µg per administration; schedules vary from single to repeated injections, administered daily or weekly.
• Autoimmunity & Treg Expansion (e.g., naïve foxp3gfp mice): Doses of 0.25 mg (250 µg) per mouse intraperitoneally for 3–7 consecutive days are common to measure effects on regulatory T cells and memory T cell expansion.
• Tumor Models (e.g., CT26, A20, MC38):
  • CT26 tumor model: 100 µg per mouse twice weekly.
  • A20 tumor model: Efficacy shown in the range of 10–30 µg per mouse biweekly when used in multi-antibody combinations; higher doses (above 30 µg per injection) were linked to increased toxicity in some contexts.
  • MC38 dual-tumor model: Similar combination therapy regimens use 10–30 µg per mouse, up to five or six injections.
• Toxicity Considerations: Adverse effects (such as anaphylaxis or neutropenia) are rare but observed, particularly with repeated or higher dosing. At 30 µg doses, some mild-to-moderate neutropenia and eosinophilia were noted, while 10 µg regimens were effective with minimal toxicity.

• Administration Route: Intraperitoneal (i.p.) is most common.
• Adjustments: Dose and frequency are commonly tailored based on:
  • Mouse strain/susceptibility
  • Immunological endpoints measured
  • Combination with other immunotherapies
  • Desired balance between efficacy and minimization of toxicity

Overall, while the 0.2–0.25 mg i.p. daily for several days regimen is frequent for T cell modulation studies, lower doses (10–30 µg) are often used—especially in combination therapies in tumor models—to reduce toxicity.