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

Use of Clone B8.2C12 in In Vivo Mouse Studies

Clone B8.2C12 is a rat monoclonal antibody that specifically recognizes mouse TIM-3 (CD366), a checkpoint protein involved in immune regulation. Its main application in vivo is to block the function of TIM-3 on immune cells, particularly in the context of tumor immunology and T cell exhaustion studies.

Specific In Vivo Experimental Applications

• Antibody Administration: Mice are injected intraperitoneally (i.p.) with B8.2C12 (typically 200 ?g per dose), often alongside isotype controls for comparison.
• Frequency and Duration: In tumor studies, the antibody is administered every third day for a total of two doses, with tumors and immune cells analyzed 48–72 hours after the last injection.
• Mouse Strains: Notably, B8.2C12 binds specifically to the BALB/c allele of TIM-3 and does not recognize the C57BL/6 allele, so its use is restricted to BALB/c mice or engineered models expressing this allele.
• Functional Blockade: The antibody does not deplete TIM-3-expressing cells but instead blocks TIM-3's interaction with its ligands, phosphatidylserine (PtdSer) and CEACAM1, disrupting downstream signaling. This blockade can be monitored using flow cytometry to assess changes in immune cell populations and TIM-3 surface expression.
• Tumor Models: In preclinical cancer studies, BALB/c mice bearing CT26 tumors (a BALB/c-derived colon carcinoma) have been treated with B8.2C12 to investigate the impact of TIM-3 blockade on tumor growth, immune infiltration, and T cell function.
• Flow Cytometric Analysis: After in vivo treatment, organs like spleen or tumor are harvested, and single-cell suspensions are stained with additional antibodies (e.g., CD4, CD8) and non-competing anti-TIM-3 clones to assess the frequency and phenotype of immune subsets by flow cytometry.

Mechanism and Epitope Specificity

• Epitope Recognition: B8.2C12 recognizes a region on the BC-loop of mTIM-3, which is polymorphic between mouse strains. This specificity underlies its selective reactivity with the BALB/c allele and distinguishes it from other anti-TIM-3 clones like RMT3-23 and 5D12, which recognize different epitopes and may exhibit broader strain reactivity.
• Functional Outcomes: The blockade of TIM-3 by B8.2C12 has been shown to modulate immune responses, particularly in models of chronic viral infection and cancer, where TIM-3 is a marker of T cell exhaustion.

Practical Considerations

• Antibody Format: B8.2C12 is available in various formats, including purified (for in vivo use) and fluorescently conjugated (for ex vivo analysis).
• Dosing and Handling: In vivo studies use purified, endotoxin-tested antibody to avoid confounding immune activation.
• Limitations: Researchers must carefully select mouse strains and confirm TIM-3 allele compatibility, as B8.2C12 is not functional in C57BL/6 mice.

Summary Table: Key Features of B8.2C12 in In Vivo Studies

Conclusion

Clone B8.2C12 is a crucial tool for in vivo studies of TIM-3 biology in BALB/c mouse models, offering a selective and functional blockade of this immune checkpoint without depleting TIM-3+ cells. Its use has advanced understanding of TIM-3’s role in cancer immunity and T cell exhaustion, but researchers must carefully consider strain compatibility and experimental design when incorporating B8.2C12 into their studies.

Based on the product specifications for clone B8.2C12, the correct storage temperature depends on the specific formulation and intended storage duration.

Standard Storage Conditions

For most B8.2C12 antibody formulations, the standard storage temperature is 2°C to 8°C (refrigerated conditions). The antibody solution should be stored undiluted at this temperature range and protected from prolonged exposure to light.

Important Storage Guidelines

Do not freeze the antibody solution, as freezing can damage the protein structure and reduce antibody functionality. The antibodies should be stored in their original undiluted form to maintain stability and activity.

Extended Storage Options

For longer-term storage of functional grade versions, the antibody can be stored at ? -70°C (-70°C or colder). However, this requires specific handling procedures:

• Aseptically aliquot the antibody into working volumes without diluting
• Avoid repeated freeze-thaw cycles, which can degrade the antibody

Short-term Storage

For immediate use, functional grade B8.2C12 antibodies may be stored at the standard 2-8°C range for up to one month.

The key is to maintain the cold chain at 2-8°C for routine storage while protecting from light exposure, and only use ultra-low temperature storage (-70°C or below) for long-term preservation when properly aliquoted to avoid freeze-thaw damage.

The antibody B8.2C12 (anti-mouse TIM-3) is frequently used in combination with other antibodies and proteins to provide mechanistic context or to enable immunophenotyping, especially in studies of T cell exhaustion, cancer immunotherapy, and immune checkpoint blockade. Some of the most commonly used antibodies and proteins in the literature with B8.2C12 include:

• Anti-PD-1 and Anti-PD-L1 antibodies: Co-inhibition (using, for example, anti-PD-1 with anti-TIM-3/B8.2C12) is widely investigated to overcome immune suppression in tumor models.
• RMT3-23 and 5D12 clones (other anti-TIM-3 antibodies): These are alternative anti-TIM-3 clones used in competition and cross-blocking studies to determine epitope specificity and functional overlap with B8.2C12.
• Anti-CD8, anti-CD4, and anti-CD3 antibodies: Commonly used for T cell subset identification or depletion and functional assays when profiling the effect of TIM-3 blockade.
• Interferon-? (IFN?) detection antibodies: Since TIM-3 is highly expressed on IFN?-producing cells, studies of immune exhaustion states and activation signatures often include these markers.
• Anti-galectin-9 antibodies or galectin-9 protein: Galectin-9 is a well-established ligand for TIM-3, and antagonism/agonism studies often combine B8.2C12 with reagents targeting galectin-9 to dissect pathway interactions.
• Anti-CEACAM1 antibodies: CEACAM1 is another ligand for TIM-3; thus, blocking studies may employ both anti-TIM-3 and anti-CEACAM1 antibodies.
• Anti-FoxP3, anti-CD25, and anti-CD44: These markers are sometimes used alongside B8.2C12 for deeper immunophenotyping of exhausted or regulatory T cells in various immune contexts (inferred from common protocols for immunophenotyping exhausted or regulatory T cell compartments in conjunction with checkpoint analysis).

In addition to these:

• Isotype controls (typically rat IgG1 ? for B8.2C12)
• Relevant cytokines (e.g., IL-2, TNF?) detection antibodies for functional profiling of T cell responses,

These reagents help delineate the immune phenotypes and mechanisms impacted by TIM-3 blockade in preclinical models.

Summary Table

These combinations reflect the current standard approaches in TIM-3 biology and immunotherapy research, as described in the referenced studies.

Clone B8.2C12 is a rat IgG1 monoclonal antibody that targets mouse CD366 (Tim-3) and has been extensively studied in the scientific literature. The key findings from research involving this antibody clone reveal several important characteristics and applications.

Epitope Recognition and Binding Specificity

B8.2C12 recognizes a unique, non-overlapping epitope on Tim-3 compared to other commonly used anti-Tim-3 antibodies. Cross-blocking experiments demonstrated that B8.2C12 does not compete for binding with other anti-murine Tim-3 antibodies such as RMT3-23 and 5D12, indicating these antibodies bind to distinct regions on the Tim-3 protein. This non-competitive binding pattern allows researchers to reliably track Tim-3-expressing cells in experimental settings where multiple antibody treatments are used.

Importantly, B8.2C12 exhibits strain-specific binding, as it only binds to the BALB/c allele of Tim-3 and does not recognize the C57BL/6 variant. This specificity is attributed to the fact that the B8.2C12 epitope covers residues in the BC-loop that are polymorphic between C57BL/6 and BALB/c mouse strains.

Functional Impact on Tim-3 Signaling

B8.2C12 has been shown to block critical Tim-3 interactions despite binding to an epitope that is remote from the primary ligand-binding sites. The antibody can interfere with both phosphatidylserine (PtdSer) and CEACAM1 binding to Tim-3, which is particularly noteworthy because these are key ligands involved in Tim-3's inhibitory signaling pathways.

Hydrogen-deuterium exchange mass spectrometry studies revealed that B8.2C12 binding causes allosteric protection of residues 99-107, a region in close proximity to the PtdSer binding site. This allosteric effect provides a mechanistic explanation for how the antibody can block ligand interactions even when binding to a distant epitope.

Therapeutic Applications and Immunomodulation

B8.2C12 has demonstrated immunomodulatory properties in vivo across multiple preclinical cancer models. The antibody has been used in combination immunotherapy approaches, where it contributes to enhancing anti-tumor immune responses by blocking Tim-3-mediated immune suppression.

In the context of immune checkpoint blockade therapy, studies have shown that targeting Tim-3 with antibodies like B8.2C12 can help reduce the fraction of inhibitory checkpoint receptors on CD8+ tumor-infiltrating lymphocytes. This includes decreasing the population of cells expressing multiple checkpoint receptors such as PD-1+TIM-3+, which are associated with dysfunctional T cell states.

Research Tool Applications

The strain-specific binding pattern of B8.2C12 makes it a valuable research tool for studies using BALB/c mice specifically. Researchers must be aware of this limitation when designing experiments, as the antibody will not be effective in C57BL/6 mouse models or other strains that express the non-BALB/c Tim-3 allele.

The antibody is commercially available in various fluorescent conjugates, including APC and PE formats, making it suitable for flow cytometry applications to identify and characterize Tim-3-expressing cell populations.

These findings collectively establish B8.2C12 as both a useful research tool for Tim-3 studies in appropriate mouse strains and a potential therapeutic agent for cancer immunotherapy, while highlighting the importance of understanding its strain-specific binding characteristics and unique epitope recognition pattern.