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

Clone OX-86 is an agonistic antibody targeting mouse OX40 (CD134), and in in vivo mouse studies, it is primarily used to stimulate the OX40 pathway, thereby enhancing T cell responses and modulating immune regulation.

Key applications and protocols include:

• Immune Activation and Modulation: OX-86 is used to transiently engage OX40 on T cells, promoting clonal expansion of both CD4 and CD8 effector T cells, augmenting cytokine production, and preventing immune tolerance induction.
• Regulatory T Cell (Treg) Expansion: Administration of OX-86 (typically 0.25 mg per mouse by intraperitoneal injection for 3 to 7 consecutive days) in naïve or transgenic mice results in rapid and robust expansion of CD4+Foxp3+ Tregs in lymphoid tissues and peripheral organs. This expansion is kinetic, peaking within one week post-treatment, and can be dose-dependent.
• Tumor Models: OX-86 has been shown to delay tumor growth in vivo by enhancing generation of antigen-specific effector T cells and reversing immunological tolerance, making it a tool in preclinical cancer immunotherapy models.
• Autoimmune and Diabetes Models: In NOD mice (a model for Type 1 diabetes), OX86 treatment stimulates Treg proliferation and can reduce disease incidence by modulating immune cell subsets.

Functional and pathological consequences:

• Despite robust Treg expansion, the suppressive function of these cells may be impaired after OX-86 treatment, which can contribute to autoimmune-like pathology in naïve mice, such as inflammatory infiltrates in organs.
• The antibody is also used to study memory CD4+ T cells, with higher doses and longer treatment further expanding these populations.

Experimental protocols:

• Standard doses: 0.2–0.25 mg intraperitoneally per mouse, administered either for 3 or 7 consecutive days.
• Analysis: Effects are typically assessed via FACS for Treg and memory T cell expansion, functional assays for suppressive capacity, and histological analysis for pathological changes.

In summary, OX-86 is utilized in mouse studies as a potent tool to stimulate the OX40 pathway, modulate immune cell populations, enhance effector T cell responses, and investigate the balance between activation, regulation, and pathogenesis in immune mechanisms.

Storage Temperature for Sterile Packaged Clone OX-86

Functional grade, sterile packaged anti-mouse CD134 (clone OX-86) is recommended to be stored at 2–8°C for up to one month after receipt. For longer-term storage, aseptically aliquot and store at –70°C or below. You should avoid repeated freeze-thaw cycles to maintain stability and effectiveness.

Additional Storage Guidance

• Do not freeze if the specific clone OX-86 comes conjugated to a fluorochrome (e.g., R-phycoerythrin, PE), as freezing may damage the conjugate and the product should only be stored at 2–8°C.
• If the product is unconjugated (purified), it may be stored long-term at lower temperatures (? –70°C) aseptically aliquoted.
• Follow the manufacturer’s lot-specific datasheet as storage recommendations can change or vary between product preparations and batches.

Summary Table

Always verify with the product’s Certificate of Analysis and manufacturer’s datasheet for batch-specific instructions.

Commonly Used Antibodies and Proteins with OX-86 in Literature

OX-86 is a well-known monoclonal antibody targeting mouse CD134 (OX-40, TNFRSF4) and is widely used to study T cell activation, immune checkpoint modulation, and tumor immunology in murine models. In the literature, OX-86 is often used alongside other antibodies and proteins to dissect immune responses, particularly those involving T cell subsets and co-stimulatory pathways.

Key Antibodies Used with OX-86

• Anti-CD4 and Anti-CD8 Antibodies: OX-86 targets CD134, which is expressed on activated CD4+ and CD8+ T cells. Therefore, co-staining with anti-CD4 and anti-CD8 antibodies is common for phenotyping and functional analysis of these T cell subsets in flow cytometry and immunohistochemistry experiments.
• Anti-Foxp3 Antibodies: Since CD134 is also expressed on activated regulatory T cells (Tregs), anti-Foxp3 antibodies are frequently used to identify and characterize Treg populations in studies involving OX-86.
• Anti-IFN-?, Anti-IL-2, Anti-IL-4, and Anti-IL-17A Antibodies: These are used in cytokine detection assays (ELISA, intracellular staining) to measure T cell effector functions in response to OX-86 stimulation or CD134 engagement. For example, capture and detection antibodies for IL-2, IFN-?, IL-4, and IL-17A are listed in cytokine measurement protocols in studies involving OX40/OX40L signaling.
• Isotype Control Antibodies: Rat IgG1? isotype controls are routinely used as negative controls in experiments employing OX-86 (which is rat IgG1, ?).

Proteins and Fusion Proteins

• OX40 Ligand (OX40L, TNFSF4): The natural ligand for CD134/OX40 is OX40L. OX40L-fusion proteins (e.g., MBL-OX40L, Fc-OX40L) are used to study receptor-ligand interactions, signal transduction, and T cell co-stimulation. These fusion proteins are employed in ELISA and cell-based assays to confirm the specificity and activity of OX-86 and to compare agonistic effects.
• OX40-Fc Fusion Protein: Used to coat ELISA plates for binding studies with OX40L or to detect soluble OX40 in experimental systems.
• Streptavidin-HRP and Biotinylated Detection Antibodies: Common in ELISA and flow cytometry for signal amplification and detection of cytokines or surface markers.

Additional Immune Markers

• Anti-CD25, Anti-CD44, Anti-CD62L: Often used in tandem with OX-86 to further characterize activated/memory T cell phenotypes.
• Anti-B220, Anti-NK1.1, Anti-Gr-1: Used to identify B cells, NK cells, and neutrophils, respectively, which can also express CD134 under certain conditions.

Summary Table

Conclusion

OX-86 is routinely used in combination with antibodies against key T cell markers (CD4, CD8, Foxp3), cytokine detection reagents, isotype controls, and OX40L or its fusion proteins to investigate the role of CD134/OX40 in immune responses, particularly in the contexts of T cell activation, memory formation, and tumor immunology. The choice of accompanying reagents depends on the specific experimental aims, but the above represent some of the most commonly co-employed tools in the literature.

Key Findings from Clone OX-86 Citations in Scientific Literature

OX-86 (clone) is a monoclonal antibody targeting murine CD134 (OX-40, TNFRSF4), a co-stimulatory receptor expressed primarily on activated T cells. Its use in research has elucidated several important biological roles and therapeutic implications, particularly in immunology and oncology.

Immunological Functions

• Costimulation of T Cell Responses: OX-86, as an agonistic antibody, significantly enhances the expansion and cytokine production (especially IL-2 and IFN-?) of both CD4? and CD8? T cells when combined with T cell receptor stimulation (e.g., anti-CD3). This effect is comparable to that of OX40 ligand (OX40L) fusion proteins, demonstrating its robust costimulatory activity in vitro.
• Role in Memory T Cell Formation: Engagement of CD134 by OX-40L (or OX-86) is critical for generating optimal memory T cell responses following immune activation. Depletion of CD134 in mouse models leads to reduced numbers of CD4? and CD8? T cells, highlighting its importance in sustaining the immune response.
• Direct Agonism via Fc?RIIb: OX-86 is a rat IgG1 antibody that binds preferentially to the inhibitory Fc? receptor Fc?RIIb. In contrast to mouse IgG2a antibodies (which interact more with activating Fc? receptors and may mediate depletion), OX-86’s Fc?RIIb engagement allows for direct, Fc?RIIb-mediated crosslinking and agonism of OX40 on T cells, even in the absence of CD4? T cell help in certain experimental settings.

Role in Cancer and Autoimmunity

• Tumor Suppressor Function: Activating antibodies like OX-86 against CD134 can enhance tumor growth in some models, suggesting that CD134 acts as a tumor suppressor. Its absence or blockade disrupts the immune response against tumors, indicating a complex role in carcinogenesis.
• Potential Therapeutic Target: The agonistic activity of OX-86 and related molecules makes CD134 a candidate for immunomodulatory therapies, especially in contexts where boosting T cell responses is desirable (e.g., cancer vaccines, chronic infections). However, the dual role of CD134 in both promoting and suppressing immune responses complicates its therapeutic exploitation.

Technical and Mechanistic Insights

• Antigen Distribution: CD134 is expressed on activated CD4? and CD8? T cells, regulatory T cells, B cells, NKT cells, NK cells, and neutrophils, but OX-86’s most pronounced effects are on activated T cells.
• Comparative Agonist Potency: In vivo, OX-86 induces significant but not maximal antigen-specific T cell responses compared to some OX40L fusion proteins, suggesting that oligomerization and Fc domain fusion can further enhance agonistic activity beyond what OX-86 alone achieves.
• Isotype-Specific Effects: The biological effects of anti-OX40 antibodies are influenced by their isotype, with rat IgG1 (OX-86) favoring direct agonism via Fc?RIIb, whereas mouse IgG2a isotypes may mediate depletion through stronger binding to activating Fc? receptors.

Summary Table: Key Biological Effects of OX-86

Conclusion

Clone OX-86 has been instrumental in demonstrating that CD134 (OX-40) is a critical immune checkpoint capable of modulating T cell activation, memory formation, and anti-tumor immunity. Its mechanism of action as a direct agonist via Fc?RIIb, alongside its complex role in cancer, positions CD134 as both a promising therapeutic target and a cautionary example of the nuanced effects of immune co-stimulation.