Anti-Mouse CD252 (OX40L) [Clone RM134L] – Purified in vivo PLATINUM™ Functional Grade

Clone RM134L is primarily used in mouse studies as a blocking antibody to investigate OX40L-OX40 signaling pathways and their roles in immune responses. This antibody specifically targets mouse OX40L (CD252), a costimulatory molecule expressed on antigen-presenting cells.

Blocking OX40/OX40L Signaling

The primary in vivo application involves using RM134L to inhibit the interaction between OX40L and its receptor OX40, which is crucial for T cell activation and proliferation. This blocking activity allows researchers to study the functional consequences of disrupting this costimulatory pathway in various disease models.

Autoimmune Disease Models

RM134L has been extensively used in experimental autoimmune encephalomyelitis (EAE) models, which mimic multiple sclerosis in humans. Treatment with RM134L ameliorated clinical signs of EAE in a dose-dependent manner in both actively induced and adoptively transferred models. The antibody worked by greatly inhibiting the accumulation of OX40-expressing CD4+ T cells and preventing the migration of pathogenic T cells into the central nervous system, though it had minimal effect on IFN-γ producing Th1 cell proliferation.

T Cell Proliferation Studies

In vivo treatment with RM134L has been demonstrated to inhibit poly(I:C)/CD40 stimulated proliferation of CD4 T cells. This application helps researchers understand how OX40L-mediated costimulation contributes to T cell expansion during immune responses.

The blocking activity of RM134L enables researchers to dissect the specific contribution of OX40-OX40L interactions in immune tolerance, T cell priming, and various pathological conditions involving excessive or inappropriate T cell activation.

RM134L, a monoclonal antibody targeting mouse CD252 (OX40L), is frequently used in combination with several other antibodies and proteins in research settings. These combinations are designed to study immune cell activation, costimulatory pathways, and inflammatory responses.

Antibodies for B Cell Activation

RM134L is commonly used alongside anti-IgM and anti-CD40 antibodies when studying B cell responses. This combination is particularly relevant because OX40L expression is induced on mouse splenic B cells when stimulated with both anti-IgM and anti-CD40 together. Neither antibody alone produces the same effect, making this a standard experimental approach for inducing OX40L expression.

OX40 Receptor-Related Reagents

OX40-Ig fusion proteins and anti-OX40 antibodies are frequently paired with RM134L in experimental designs. These reagents allow researchers to study the OX40-OX40L signaling axis from both sides of the interaction, helping to elucidate the costimulatory mechanisms between antigen-presenting cells and T cells.

T Cell Activation Markers

Anti-CD3 antibodies are commonly used with RM134L in proliferation assays. RM134L has been shown to inhibit the costimulatory activity of OX40L-transfected cells when T cells are stimulated with anti-CD3, making this combination valuable for studying T cell proliferation and activation.

Other TNF Superfamily Blocking Antibodies

In comparative studies of costimulatory molecules, RM134L is used alongside blocking antibodies for other TNF superfamily members, including FR70 (anti-CD70), TKS-1 (anti-41BBL), and RM153 (anti-CD30L). These combinations help researchers understand the relative contributions of different costimulatory pathways in immune responses.

Additional Lymphocyte Markers

RM134L is also utilized with antibodies recognizing key lymphocyte markers, which allows for comprehensive phenotyping of immune cell populations in flow cytometry and functional assays, depending on the specific experimental goals and model systems being studied.

The RM134L clone, a monoclonal antibody targeting mouse CD252 (OX40L), has generated significant findings across multiple areas of immunological research, particularly in understanding costimulatory pathways and developing potential therapeutic interventions.

Therapeutic Applications in Autoimmune Disease

Research using RM134L has demonstrated substantial therapeutic potential in experimental autoimmune encephalomyelitis (EAE), an animal model for multiple sclerosis. When Nohara and colleagues administered RM134L to mice with EAE induced by PLP 139-151 peptide, the treatment ameliorated clinical signs in a dose-dependent manner in both actively induced and adoptively transferred EAE models. The antibody greatly inhibited the accumulation of OX40-expressing CD4+ T cells and prevented pathogenic T cell migration into the central nervous system, though it had minimal effect on IFN-γ producing Th1 cell proliferation in draining lymph nodes.

Similarly, Weinberg and colleagues achieved clinical improvement in EAE by blocking OX40-OX40L engagement, observing 50-70% inhibition of T cell proliferation and marked reduction in disease severity with quicker recovery times. However, the frequency and severity of relapses remained unchanged, suggesting the therapeutic effect may be short-lived.

Mechanisms of T Cell Regulation

The RM134L antibody has been instrumental in elucidating how OX40-OX40L signaling regulates T cell responses. Studies revealed that OX40 signaling provides essential costimulatory signals to antigen-reacting naive T cells, prolonging proliferation and augmenting production of multiple cytokines including IL-2, IL-3, and IFN-γ. In vivo treatment with RM134L has been shown to inhibit poly(I:C)/CD40-stimulated proliferation of CD4 T cells.

Adoptive transfer experiments using RM134L provided critical insights into disease pathogenesis: wild-type donor T cells efficiently transferred EAE disease to OX40L-deficient mice, while OX40L-deficient donor T cells failed to transfer disease to wild-type recipients. This demonstrated that OX40-OX40L interaction plays a crucial role in the T cell priming process during EAE development.

Functional Characterization of OX40L Expression

The RM134L clone has been essential for mapping OX40L expression patterns across immune cell populations. Research confirmed that this antibody effectively stains B cells activated for four days with anti-IgM plus anti-CD40 antibodies. While OX40-Ig fusion protein studies suggested OX40L expression on LPS-activated splenic B cells and activated CD4+/CD8+ T cells, the RM134L antibody could not confirm these findings, highlighting important technical considerations in detecting this costimulatory molecule.

Blocking Activity and Mechanistic Studies

Functionally, RM134L effectively inhibits the binding of OX40-Ig fusion protein to OX40L transfectants and blocks the costimulatory activity of OX40L. This blocking capability has made it valuable for dissecting the specific contribution of OX40-OX40L interactions in various immune responses, from protective immunity against viral infections to pathological responses in autoimmune conditions.

The research utilizing RM134L has fundamentally advanced understanding of the OX40/OX40L costimulatory pathway and its role in both protective and pathological immune responses, establishing a foundation for potential therapeutic applications in allergic diseases, autoimmune conditions, and viral infections.

Dosing Regimens of Clone RM134L in Mouse Models

Overview
Clone RM134L is a monoclonal antibody targeting mouse OX40L (CD252), a costimulatory molecule involved in T cell activation and proliferation. Its therapeutic and experimental use in mouse models is primarily for blocking the OX40–OX40L pathway, which has been implicated in autoimmune diseases, transplant models, and potentially cancer immunotherapy.

Variability in Dosing Regimens

Dose, Timing, and Duration
Dosing regimens for RM134L are not standardized and vary significantly depending on the disease model, experimental objectives, and endpoints. The manufacturer and suppliers note that “dosing regimens vary in amount, timing, and duration depending on the mouse disease model and experimental design,” and recommend optimization for each specific application. However, published studies provide some concrete examples:

• EAE Model: In studies of autoimmune encephalomyelitis, doses of RM134L ranging from 0.1 mg to 1.0 mg per mouse were administered intraperitoneally, daily, for 7–10 days. The treatment was initiated at the onset of clinical signs, and efficacy was dose-dependent, with higher doses generally providing greater amelioration of disease symptoms.
• Flow Cytometry: For analytical (non-therapeutic) applications such as flow cytometry, typically ≤0.5 µg of antibody per test (defined as staining a cell sample in 100 µL) is used, with cell numbers empirically determined but ranging from (10^5) to (10^8) per test.
• Other Models: Suppliers and manufacturers emphasize that dosing must be optimized for each experimental setup. No single regimen is universally applicable, and timing (prophylactic vs. therapeutic), frequency (single vs. multiple doses), and route (intraperitoneal, intravenous, or other) are adjusted based on the model and desired outcome.

Considerations for Experimental Design

• Model-Specific Optimization: The biological context (e.g., autoimmune, transplant, tumor) and the stage of disease (induction, progression, remission) heavily influence the optimal regimen.
• Endpoint Assessment: The choice of dose and schedule should be guided by the primary endpoint, whether it is disease severity, survival, immune cell infiltration, or biomarker expression.
• Combination Therapy: Some studies use RM134L in combination with other immunomodulatory agents, which may require further adjustment of dosing to avoid toxicity or achieve synergy.

Summary

There is no universal dosing regimen for clone RM134L across mouse models. In the EAE model, doses of 0.1–1.0 mg/mouse, given i.p. daily for 7–10 days, have shown efficacy. For flow cytometry, ≤0.5 µg per test is standard. In all cases, careful titration and model-specific optimization are essential, and researchers should consult both supplier recommendations and published protocols for their particular application.