Anti-Mouse CD366 (Tim-3) [Clone RMT3-23] — Purified in vivo GOLD™ Functional Grade

Clone RMT3-23 is a rat anti-mouse monoclonal antibody commonly used to functionally block the TIM-3 (CD366) immune checkpoint receptor in in vivo mouse studies, particularly to investigate the role of TIM-3 in immune regulation and tumor immunity.

Key in vivo applications and mechanisms include:

• Antagonist/blocking antibody: RMT3-23 is used to block TIM-3 receptor signaling on T cells (and other immune cells), thereby preventing its inhibitory effects on anti-tumor and inflammatory responses.
• Tumor immunology: Studies have used RMT3-23 as a monotherapy or in combination with other immune modulators (such as anti-PD-1 or adenosine 2A receptor antagonists) to demonstrate slowing of tumor growth by enhancing cytolytic activity of immune cells in tumor-bearing mice.
• Autoimmunity and tolerance: RMT3-23 is employed in models of inflammatory diseases—such as experimental autoimmune encephalomyelitis (EAE), type 1 diabetes, and graft-versus-host disease (GVHD)—where it is used to study how TIM-3 contributes to immunological tolerance or the control of Th1-mediated inflammation.
• Mechanistic studies: Administration of RMT3-23 in vivo does not deplete TIM-3+ cells, but can cause down-modulation (internalization or surface reduction) of TIM-3 expression on these cells. This effect often requires cross-linking of the antibody.
• Functional inhibition: RMT3-23 has been shown to block interaction of TIM-3 with its ligand, galectin-9, which is important for the induction of cell death in Th1 cells and regulation of immune responses.

Principal cell types targeted:

• Effector T cells (Th1 and CD8+ Tc1)
• Regulatory T cells (Tregs)
• Macrophages and myeloid cells
• NK cells and mast cells

When used for in vivo studies (e.g., via intraperitoneal or intravenous injection), RMT3-23 can probe the function of TIM-3 in:

• Anti-tumor immune responses and resistance mechanisms
• Therapies designed to overcome immune exhaustion or checkpoint inhibition
• Pathogenesis and treatment of autoimmune and inflammatory disorders

RMT3-23 is widely cited as the standard blocking antibody for murine TIM-3 studies in both basic and preclinical translational research.

The correct storage temperature for sterile packaged clone RMT3-23 (anti-mouse CD366/TIM-3 antibody) is 2–8°C for short-term storage (up to one week), typically in a refrigerator and protected from light, undiluted, and do not freeze. For long-term storage, you should aliquot the antibody and store it at -20°C in a manual defrost freezer, while avoiding repeated freeze-thaw cycles. Always refer to the lot-specific datasheet, as formulations may occasionally differ depending on the vendor or conjugation.

Key points:

• Short-term storage: 2–8°C (refrigerator), do not freeze, store undiluted, protect from light.
• Long-term storage: Aliquot and store at -20°C (manual defrost freezer), avoid freeze-thaw cycles.
• Do not store at room temperature or in frost-free freezers.
• Always check the manufacturer's datasheet for specific handling instructions for your batch.

Commonly used antibodies or proteins with RMT3-23 (an anti-mouse TIM-3/CD366 antibody) in the literature include other immune checkpoint inhibitors, especially those targeting PD-1 and PD-L1, as well as alternative anti-Tim-3 clones and proteins interacting with the TIM-3 pathway.

Key antibodies and proteins commonly used alongside RMT3-23:

• Anti-PD-1 or anti-PD-L1 antibodies: RMT3-23 is frequently combined with antibodies blocking PD-1 or PD-L1 to investigate synergistic effects on anti-tumor immunity and T cell exhaustion in various cancer models.
• Other anti-TIM-3 clones: Notable clones include:
  • 5D12 (mouse IgG1)
  • B8.2C12 (rat IgG1)These are used for comparative studies or in combination to block different TIM-3 epitopes.
• Anti-CD4, Anti-CD8, and other lineage markers: These antibodies are used to phenotype and sort T cell populations for mechanistic studies of TIM-3-mediated immune regulation.
• Galectin-9: As a known ligand for TIM-3, recombinant galectin-9 protein is often used in binding and blockade assays to assess the effects of RMT3-23 and related antibodies on TIM-3 signaling.
• CEACAM1 and phosphatidylserine (PtdSer): Proteins used in ligand-binding studies to test the blocking efficiency of different TIM-3 antibodies, including RMT3-23.

Summary table:

These combinations are most relevant in the context of cancer immunotherapy and functional dissection of T cell exhaustion and immunoregulation.

Clone RMT3-23 is a widely used rat IgG2a monoclonal antibody that targets murine Tim-3, a key immune checkpoint protein involved in T cell regulation and cancer immunotherapy research. Its major scientific findings are summarized as follows:

• Epitope Specificity: RMT3-23 binds a unique, non-overlapping epitope on mouse Tim-3 compared to other anti-Tim-3 clones, allowing researchers to track Tim-3-expressing cells in vivo without interference from other Tim-3 antibodies.
• Functionality In Vivo: RMT3-23 does not deplete Tim-3^+^ T cells when administered to mice, but it can cause down-modulation of Tim-3 surface expression if cross-linked, distinguishing its mechanism of action from potential depleting antibodies.
• Immunomodulatory Role: Studies have shown monotherapeutic efficacy of RMT3-23 in preclinical models, including certain sarcomas, indicating its potential to modulate immune responses against tumors.
• Applications: RMT3-23 is validated for immunohistochemistry, especially in CODEX® tissue imaging platforms for mouse models such as T-cell sarcoma. It detects Tim-3 on a range of cells: activated CD4^+^ Th1, CD8^+^ Tc1, regulatory T cells (Tregs), CD11b^+^ macrophages, NK cells, and mast cells.
• Disease Modeling: Tim-3, as detected by RMT3-23, is implicated in limiting Th1-mediated inflammatory diseases (e.g., EAE, IBD, type I diabetes) and inducing immunological tolerance via apoptosis or inhibition of effector T cell function.

Supporting Details:

• RMT3-23 does not deplete T cells, but can reduce (down-modulate) Tim-3 surface protein intensity under specific conditions, an important consideration for interpreting functional outcomes in experiments.
• This clone enables reliable detection of Tim-3, supporting the study of immune cell distribution and tumor microenvironment analysis without cross-reactivity from other clones.

Additional Notes:

• Compared to other research antibodies (like B8.2C12 and 5D12), RMT3-23 offers distinct specificity and a unique mechanism—modulating, but not depleting, Tim-3-expressing cells.
• RMT3-23 is formulated for compatibility with high-resolution multiplex imaging, enhancing its utility for advanced tissue and tumor immune microenvironment studies.

In summary, clone RMT3-23 is a highly specific, non-depleting antibody for murine Tim-3, instrumental in cancer immunotherapy and inflammation research, with unique epitope recognition and validated applications in multiplex tissue imaging.