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

Clone B8.2C12 is primarily used in in vivo mouse studies to block mouse Tim-3 (CD366) function, particularly in BALB/c mice, and is applied in tumor models and immunological modulation experiments. In these studies, B8.2C12 does not deplete Tim-3^+^ cells but instead interferes with Tim-3's interaction with its ligands and modulates immune activity.

Key uses and experimental details:

• Allele specificity: B8.2C12 binds strongly to the Tim-3 allele in BALB/c mice, but shows weak or no binding to the C57BL/6 allele.
• Blocking Tim-3 function: This antibody is used to block Tim-3 interactions with ligands such as phosphatidylserine and CEACAM1, despite having its epitope remote from the direct binding sites. It exerts its effect through allosteric protection of nearby residues.
• Non-depleting action: B8.2C12 does not deplete Tim-3-expressing cells in vivo (i.e., no significant reduction in CD4^+^ or CD8^+^ T cell numbers following administration), making it suitable for functional blockade studies rather than cell depletion assays.
• Immunotherapy and tumor models: In studies involving BALB/c mice implanted with CT26 tumors (a colon carcinoma line), mice bearing established tumors receive intraperitoneal injections of 200 ?g B8.2C12 every third day (total of two doses). Tumors and immune cells are analyzed 48–72 hours after the last injection for changes in immune activity and tumor response.
• Flow cytometry and immunophenotyping: B8.2C12 is used for flow cytometric staining to detect Tim-3 expression or monitor modulation after antibody treatment, often in combination with other immune markers.
• Functionality: B8.2C12 is categorized as a functional/blocking antibody and is suitable for in vivo immune checkpoint studies, especially investigating Tim-3's role in T cell regulation, exhaustion, or tumor immune escape.

Experimental considerations:

• Strain specificity is crucial: Because B8.2C12 does not effectively bind Tim-3 in C57BL/6 mice, it is not appropriate for studies using this strain.
• Dosing and administration: The typical protocol is intraperitoneal injection of 200 ?g per mouse every third day for two doses.
• Controls: Isotype-matched controls and non-competing anti-Tim-3 antibodies are often used to assess specificity and immune modulation.

Summary Table: B8.2C12 Use in Mouse Models

In summary, clone B8.2C12 is an allele-specific, non-depleting, blocking antibody widely employed in BALB/c mouse studies to investigate Tim-3's immunoregulatory functions, particularly in tumor and checkpoint blockade models.

Commonly used antibodies or proteins with B8.2C12 (an anti-mouse TIM-3 antibody) in the literature include other checkpoint inhibitors, T cell markers, and relevant immunoregulators for both mechanistic and combinatorial studies.

Key co-used antibodies and proteins include:

• Anti-PD-1 (clone J43): Frequently combined with B8.2C12 to investigate synergistic effects on immune responses, particularly in tumor models.
• Anti-KLRG-1 (clone 2F1): Used in flow cytometry panels to delineate T cell subsets.
• Anti-CD4 and Anti-CD8: Standard markers to identify major T cell populations when measuring the effects of TIM-3 blockade.
• Anti-TIM-3 clones (RMT3-23 and 5D12): Used to compare binding properties, competition, and in multi-antibody experiments for robust detection of TIM-3 or mechanistic elucidation.
• Other checkpoint inhibitors: Such as anti-PD-L1; commonly used in combination with B8.2C12 to study co-inhibition and assess combinatorial therapeutic approaches.
• Anti-galectin-9: Since galectin-9 is a known ligand of TIM-3, its detection or blockade may be used in conjunction with B8.2C12 to assess functional TIM-3-ligand interactions.
• Cytokines and stimulatory antibodies (anti-CD3, anti-CD28): Used in context of activation studies, to evaluate T cell function under TIM-3 modulation.

These markers and antibodies are typically integrated in flow cytometry panels and functional assays to define cell subsets (e.g., CD4^+, CD8^+ T cells), characterize exhaustion or activation (e.g., KLRG-1, PD-1), and dissect checkpoint signal pathways.

Summary table:

These combinatorial approaches enable detailed cellular and functional immune profiling in studies utilizing B8.2C12.

Key findings regarding clone B8.2C12 from scientific literature are as follows:

• Clone B8.2C12 is a rat monoclonal IgG1 antibody specific for murine Tim-3 (CD366). It is widely used to study Tim-3’s function and cell expression in mouse models.

• Epitope specificity and binding profile:

  • B8.2C12 binds a unique, non-overlapping epitope on the Tim-3 protein compared to other common anti-Tim-3 clones (RMT3-23 and 5D12), as demonstrated by cross-blocking experiments. This is crucial for tracking Tim-3-expressing cells or conducting combination blockade experiments.
  • Its epitope encompasses the BC-loop region, which is polymorphic between mouse strains BALB/c and C57BL/6, explaining its allele-specific binding: B8.2C12 only binds the BALB/c Tim-3 allele.

• Functional impact:

  • B8.2C12 can block Tim-3 interactions with its ligands, particularly phosphatidylserine (PtdSer) and CEACAM1, not by direct competition but likely through allosteric protection of residues proximal to the binding site.
  • Important for immunomodulatory studies, particularly in cancer and infectious disease models, where Tim-3’s role as a checkpoint receptor on T cells is being investigated.

• Application notes:

  • It is primarily validated for flow cytometry staining of BALB/c mouse cells and researchers should be cautious when selecting it for experiments involving different mouse strains.
  • Typical usage is ?0.25 µg per million cells in flow cytometric analyses.

• Cited use cases:

  • Studies using B8.2C12 have explored its effect on dendritic cell populations, T cell exhaustion, and checkpoint blockade in mouse models, especially when allele specificity is needed or advantageous.

In summary, the key literature findings highlight B8.2C12’s unique epitope, allele-specific binding, and utility for functional blocking of Tim-3 in BALB/c mice, supporting its continued use in mechanistic and immunological studies.

Dosing regimens for clone B8.2C12, an anti-mouse Tim-3 antibody, primarily depend on application (e.g., flow cytometry vs. in vivo blocking) and the genetic background of the mouse model, with important allele-specific binding considerations.

Key points on dosing regimens across mouse models:

• Flow Cytometry (Ex Vivo Studies):

  • Recommended dose: ?0.25?µg per million cells in 100?µl volume for staining.
  • Mouse strain specificity: B8.2C12 binds ONLY the BALB/c allele of Tim-3—it does not cross-react with all mouse strains.
  • Titration: It is suggested to titrate for each experiment to optimize performance.

• In Vivo Experiments:

  • Published in vivo use: In one study, B8.2C12 was administered intraperitoneally (i.p.) to Tim-3 transgenic mice.
  • Exact dose and frequency: Not explicitly stated in the cited study; however, the protocol involved single or repeated i.p. injections with analysis after 48?h to assess immune cell changes.

• Comparison with Other Anti-Tim-3 Clones:

  • Many in vivo studies (especially in C57BL/6 and derived strains) use alternative clones such as RMT3-23, typically at 200µg per injection i.p. every 5 days for functional blocking.
  • The B8.2C12 clone’s use is limited by its allele specificity, so its in vivo application is restricted largely to BALB/c or Tim-3 transgenic models expressing the relevant allele.

• Summary Table: B8.2C12 Dosing by Mouse Model

Supporting context:

• The strict allele specificity (BALB/c) mandates careful strain selection when planning in vivo interventions with B8.2C12.
• For in vivo blocking or depletion studies in other mouse strains (e.g., C57BL/6), use of clone B8.2C12 is not appropriate; opt for broader-reacting clones like RMT3-23.

If you need details for a specific experimental setup or mouse strain, or seek non-flow-cytometry protocols, precise published dosing regimens for B8.2C12 may require consultation of primary literature or contacting antibody suppliers for additional technical guidance.