Anti-Mouse TIM-3 (Clone B8.2C12) – Purified in vivo PLATINUM™ Functional Grade

Clone B8.2C12 is a rat monoclonal antibody against mouse TIM-3 (CD366) that is widely used in murine immunology research, but its binding is specific to the BALB/c allele of TIM-3 (with significantly weaker reactivity to the C57BL/6 allele).

The most common in vivo applications in mice for clone B8.2C12 include:

• Immune checkpoint blockade studies:
  Used as an inhibitory (blocking) antibody to block TIM-3 signaling. This enables researchers to investigate the functional role of TIM-3 in regulating immune responses, particularly T cell exhaustion, tolerance, and checkpoint inhibition in cancer and autoimmunity models.

• Studies of T cell-mediated immunity such as Th1 regulation and CD8+ T cell function:
  Since TIM-3 is highly expressed on Th1 lymphocytes and certain activated CD8+ T cells, B8.2C12 is used to study the functional consequences of TIM-3 blockade on cytokine production (e.g., IFN-γ) and effector T cell proliferation.

• Analysis of antigen-specific tolerance:
  B8.2C12 has been used in models addressing the role of TIM-3 in peripheral tolerance, showing that blocking TIM-3 can disrupt antigen-specific tolerance induction and exacerbate models of autoimmune disease (such as experimental autoimmune encephalomyelitis).

• Cancer immunotherapy models:
  TIM-3 is a relevant checkpoint in tumor immunology. B8.2C12 is used in tumor-bearing mice (mainly on a BALB/c background) to test the effect of TIM-3 blockade on anti-tumor immunity, often in combination with other checkpoint inhibitors (such as anti-PD-1) to evaluate synergy.

Important technical note:

• Clone B8.2C12 is not suitable for use in C57BL/6 mice or other strains that do not express the BALB/c allele, due to poor or absent binding.
• Applications are typically in vivo blocking/functional studies and flow cytometric phenotyping (when cells are isolated from treated animals).

Summary Table: Common In Vivo Applications of Clone B8.2C12 in Mice

There are other clones (such as RMT3-23) that do not exhibit this strain specificity and may be more appropriate for C57BL/6 mice.

When designing experiments, it is essential to ensure BALB/c or compatible strains are used for in vivo studies with B8.2C12.

Based on the available information, B8.2C12 is commonly used alongside several other antibodies and proteins in research contexts, particularly in studies involving TIM-3 (CD366) immune checkpoint signaling.

Other Anti-TIM-3 Antibody Clones

The most frequently mentioned companion antibodies to B8.2C12 are RMT3-23 and 5D12, which are different anti-TIM-3 clones used for comparative studies. These three clones differ in their species origin and isotype characteristics: B8.2C12 is a rat IgG1, RMT3-23 is a rat IgG2a, and 5D12 is a mouse IgG1. Notably, while B8.2C12 binds specifically to the BALB/c allele of TIM-3 with significantly weaker reactivity to the C57Bl/6 allele, RMT3-23 can efficiently bind to both BALB/c and C57BL/6 TIM-3. These clones are often used in combination to block different epitopes or compared to evaluate their relative efficacy in functional studies.

Co-Checkpoint Inhibitors

B8.2C12 is studied in conjunction with other immune checkpoint proteins, particularly PD-1. Research has shown that CD8 T cells expressing both TIM-3 and PD-1 exhibit greater defects in cell-cycle progression and effector cytokine production compared to cells expressing PD-1 alone. This suggests that B8.2C12 may be used in experiments examining dual checkpoint blockade strategies.

TIM-3 Ligands and Binding Partners

B8.2C12 is used to study TIM-3 interactions with several key binding partners, including galectin-9 (GAL-9), phosphatidylserine (PtdSer), HMGB1, and CEACAM1. These ligands activate various biochemical pathways such as immune tolerance, T cell depletion, NF-κB signaling, and IL-2 secretion.

Key findings from scientific literature on clone B8.2C12 focus on its specificity as an anti-mouse Tim-3 antibody, its restricted allele binding, and its applications in immunological research:

• Epitope specificity and mechanism: B8.2C12 recognizes an epitope in the BC-loop region of mouse Tim-3, which is polymorphic between different mouse strains. This means B8.2C12 shows selective binding based on Tim-3 allelic variation.

• Allele specificity: B8.2C12 binds only to the BALB/c allele of Tim-3, and not to the C57BL/6 allele. This is a key consideration when choosing mouse models for experiments, as the antibody is ineffective for any strain other than BALB/c.

• Epitope location, competition, and blocking function:

  • The B8.2C12 epitope on Tim-3 does not overlap with RMT3-23 or 5D12 clones, supporting use in non-redundant or combination studies.
  • Although its binding site is remote from the primary ligand-binding site, B8.2C12 can block Tim-3 interactions with CEACAM1 and phosphatidylserine (PtdSer) through allosteric effects.
  • The BC-loop region it binds to is close to functionally important domains, explaining its blocking properties.

• Research and diagnostic application:

  • B8.2C12 is used to identify and isolate Th1 cells, as it was generated using Th1-polarized T cells and specifically stains Th1 versus Th2 cells.
  • Widely employed for flow cytometry and immunophenotyping to detect Tim-3, especially in BALB/c-derived T cells and tumor immunology studies.
  • Utilized in research on T cell exhaustion, tumor immunology, and as part of checkpoint blockade investigations due to its ability to modulate and mark Tim-3 expression.

• Functional relevance:

  • Tim-3, detected by B8.2C12, marks terminally differentiated and exhausted T cells in tumors and chronic infections.
  • The antibody aids in studying immune checkpoints, given Tim-3’s role in T cell inhibition and tumor immune evasion.

• Limitations:

  • Allele restriction is a significant limitation; it cannot be used in C57BL/6 mice and other strains not expressing the BALB/c Tim-3 allele.

Summary Table: Clone B8.2C12 Findings

These findings highlight B8.2C12’s high allele specificity, utility in immunophenotyping, flow cytometry, and checkpoint blockade research, but also emphasize careful strain selection due to its restricted binding profile.

Dosing regimens for clone B8.2C12, an anti-mouse TIM-3 antibody, can vary across different mouse models depending on several factors, including the mouse strain, age, and disease/model system being studied. Here are some general considerations and specific uses:

General Considerations for Dosing Regimens

• Mouse Strain and Age: The dosing regimen may need to be adjusted based on the specific mouse strain and age used in the study. For example, BALB/c mice are typically used with clone B8.2C12 because it only binds to the BALB/c allele of Tim-3.
• Disease/Model System: The type of disease or model system (e.g., cancer, autoimmune diseases) can influence the dosing regimen. For instance, in cancer models, the timing and frequency of dosing may be tailored to optimize antitumor effects.
• Route of Administration: Intraperitoneal (i.p.) injection is a common route for administering antibodies like B8.2C12 in mouse models.

Specific Applications

• Cancer Models: In studies involving cancer, B8.2C12 may be used in combination with other checkpoint inhibitors to enhance antitumor immunity. However, specific dosing regimens for B8.2C12 in cancer models are not detailed in the provided sources.
• Immune Studies: In immune-related studies, B8.2C12 is often used to analyze Tim-3 expression without depleting Tim-3+ cells. The dosing regimen may focus on achieving optimal staining or blocking effects rather than cell depletion.

Dosing Regimen Example

While specific dosing details for B8.2C12 are not widely reported, other antibodies like RMT3-23, another anti-Tim-3 clone, are administered at dosages around 200 µg per mouse via i.p. injection, repeated every few days. The exact dosing for B8.2C12 would require titration to ensure optimal performance for each specific application.