Anti-Mouse TIM-1 (CD365) [Clone RMT1-10] — Purified in vivo GOLD™ Functional Grade

RMT1-10 is a rat anti-mouse TIM-1 monoclonal antibody (IgG2a) that has been extensively used in various in vivo mouse studies to modulate immune responses and investigate therapeutic applications. The clone demonstrates significant biological effects across multiple disease models.

Administration and Dosing Protocols

RMT1-10 is typically administered through prolonged treatment regimens rather than single-dose protocols. In atherosclerosis studies, the antibody was given every other day for 8 weeks while ApoE-knockout mice were fed a high-fat diet. For transplantation studies, the antibody was administered to recipient mice to evaluate its effects on allograft survival.

Atherosclerosis Research Applications

In atherosclerosis models, RMT1-10 treatment produces remarkable therapeutic effects by selectively expanding atheroprotective B1a cell populations. The treatment doubles the number of peritoneal B1a cells and increases TIM-1 expression on these cells from 40% to 62%. This selective expansion results in a nearly 3-fold increase in peritoneal TIM-1+ B1a cells, which are crucial for producing protective IgM antibodies. The therapeutic approach shows promise for treating established atherosclerosis by retarding disease progression through this B1a cell-mediated mechanism.

Transplantation and Immunology Studies

RMT1-10 has proven highly effective in organ transplantation models, significantly prolonging allograft survival in both cardiac and skin transplant studies. In cardiac transplantation experiments using B6 hearts transplanted into BALB/c recipients, RMT1-10 treatment resulted in significant prolongation of allograft survival. The antibody works by altering the balance between effector T cells (Teffs) and regulatory T cells (Tregs), creating a more tolerogenic immune environment.

In skin transplantation models using the bm12 into B6 MHC class II-mismatched system, RMT1-10 extended median survival time from 13 days in untreated controls to 46.5 days in treated animals. Importantly, this protective effect was dependent on the presence of CD4+CD25+ regulatory T cells, as their depletion with anti-CD25 antibody completely abrogated the graft-protective effects of RMT1-10.

Immunomodulatory Mechanisms

The clone functions as both a stimulatory and modulatory agent depending on the experimental context. RMT1-10 binding to TIM-1 results in dramatic shifts in cytokine production profiles. In transplantation studies, treatment significantly reduced the frequency of allospecific IFN-?-producing T cells (from 226.2 ± 49.4 to 42.20 ± 12.6) while preserving IL-5-producing Th2 cells. This represents a fundamental shift from Th1-mediated rejection responses toward more tolerogenic Th2 responses.

In cell culture applications that complement in vivo studies, blocking TIM-1 with RMT1-10 reduces T cell proliferation, inhibits IFN-? and IL-17 production, and induces Th2 cytokines IL-4 and IL-10. This dual functionality makes it valuable for understanding TIM-1's role in immune regulation.

Therapeutic Potential and Clinical Implications

The consistent effectiveness of RMT1-10 across different disease models suggests significant therapeutic potential. In atherosclerosis research, antibody-mediated in vivo expansion of atheroprotective B1a cells represents a promising approach for treating patients with established atherosclerosis to prevent related vascular disorders such as heart attacks and strokes. The ability to selectively expand beneficial immune cell populations while modulating harmful inflammatory responses positions RMT1-10 as a valuable tool for developing targeted immunotherapies.

Based on the specifications for the RMT1-10 clone antibody, the correct storage temperature depends on the duration of storage needed.

Short-term Storage

For short-term storage up to 4 weeks, the antibody should be stored at 4°C.

Long-term Storage

For long-term storage, the antibody should be stored at -20°C. This temperature maintains the antibody's stability and binding capacity without significant loss of activity.

Additional Storage Guidelines

Avoid freeze-thaw cycles as these can cause protein denaturation and decrease antibody activity. To prevent this, it's recommended to aliquot the antibody into smaller volumes so you only thaw what you need for each experiment.

Storage location is also important - keep the antibody in the back of the refrigerator or freezer where temperature fluctuations are minimal, rather than near the door. Additionally, storage in frost-free freezers is not recommended due to temperature cycling.

The RMT1-10 antibody is typically supplied as a liquid formulation in phosphate buffered saline with 0.09% sodium azide at a concentration of 1 mg/ml. Upon delivery, it should be aliquoted and stored according to these temperature guidelines to maintain optimal performance.

Researchers commonly use antibodies or proteins that mark immune cell subsets, cytokines, and TIM family proteins in conjunction with RMT1-10 (anti-TIM-1) to analyze immune regulation and disease mechanisms. The specific markers and antibodies typically include:

• CD19, CD5: To identify B1a B cells, especially during studies of atherosclerosis, autoimmune models, or flow cytometry analysis.
• IgM: Used to quantify immunoglobulin M production, a marker of atheroprotective B1a cells expanded by RMT1-10.
• IL-10: Often measured as a marker of immunomodulatory regulatory B cells (B10 cells) following RMT1-10 treatment.
• CD1d: Employed to further characterize B cell subsets such as regulatory B cells expanded/affected by RMT1-10.
• TIM-1 (other clones): Antibodies such as 3B3 and 3D10 are used to compare effects, as different clones exert opposite functions—3B3 enhances Th2 responses, while 3D10 may accelerate disease.
• Tim-4: The natural ligand for TIM-1, often relevant in functional studies on antigen-presenting cells (APCs) and their interactions with TIM-1 expressing T or B cells.
• CD4, CD8: Important for examining T cell subsets in contexts such as transplant models or immune activation, sometimes assessed alongside RMT1-10 to study shifts in Th1/Th2 or Treg populations.
• IFN-?: Used to monitor cytokine secretion changes in CD8+ T cells when studying the effects of RMT1-10 on T cell-mediated models.
• Th2 and Th17 markers: Evaluated in some studies investigating the role of TIM-1 agonist/antagonist antibodies in shifting the Th balance (e.g., pro-inflammatory Th17 responses).

These markers reveal how RMT1-10 interacts with immune cell populations, modulates cytokine production, and can shift disease outcomes, often measured by flow cytometry or immunoassay methods. Studies frequently include control antibodies (isotype controls or other anti-TIM-1 clones), specifically to delineate whether observed effects are unique to RMT1-10 or general to TIM-1 blockade.

Other possible proteins/antibodies sometimes used in multicolor flow cytometry panels or mechanistic studies include:

• CD45: General leukocyte marker.
• Foxp3: For regulatory T cell (Treg) populations in studies of immune tolerance or transplantation.
• Additional cytokine markers (IL-6, TNF-?): In studies of inflammation or immune activation.

In summary, the most commonly used antibodies and proteins with RMT1-10 are those that identify B cell, T cell, and regulatory/effector cell populations (CD19, CD5, CD1d, IgM, IL-10, CD4, CD8, Foxp3, IFN-?) and other anti-TIM-1 clones (3B3, 3D10), serving as critical controls or comparators in experimental immunology.

Key Findings from Scientific Literature on Clone RMT1-10

Immunomodulation and Atherosclerosis

• Prolonged RMT1-10 Treatment in ApoE-KO Mice: Administration of the anti-TIM-1 monoclonal antibody RMT1-10 (every other day for 8 weeks) in ApoE-deficient mice on a high-fat diet led to a significant increase in peritoneal B1a cell numbers, especially those expressing TIM-1 and IgM.
• Elevated Plasma IgM: RMT1-10 treatment increased both total plasma IgM and malondialdehyde (MDA)-oxidized LDL (oxLDL)-specific IgM by 33% and 40%, respectively, without affecting total Ig or IgG levels.
• Reduced Atherosclerotic Lesion Severity: The treatment increased lesion IgM deposition by about 170% and reduced MDA-oxLDL accumulation in lesions by nearly 30%, suggesting a protective role against atherosclerosis progression.
• Mechanism: The expansion of B1a cells and elevation of IgM appears TIM-1-dependent, as TIM-1–negative B1a cells did not increase in number, and TIM-1+IgM+ B1a cells showed the most pronounced expansion.
• No Effect on IL-10: Plasma IL-10 levels were unchanged, indicating that the atheroprotective effects were independent of systemic IL-10 modulation.

Transplantation and Immune Tolerance

• Prolonged Allograft Survival: In a fully MHC-mismatched cardiac transplant model, a short-course monotherapy with RMT1-10 significantly prolonged allograft survival compared to controls (median survival time 22 vs. 9 days).
• Th2 Bias in Immune Response: RMT1-10 treatment reduced the frequency of IFN-?-secreting (Th1) alloreactive T cells and increased IL-5-secreting (Th2) cells, shifting the immune response toward a less inflammatory, more tolerogenic profile.
• Dependence on Regulatory T Cells (Tregs): The graft-protective effect of RMT1-10 was abrogated by depletion of CD4+CD25+ Tregs, highlighting the importance of regulatory T cells in mediating the antibody’s effect.

General Immune Modulation

• Differential Effects Compared to Agonistic Antibodies: Unlike the agonistic anti-TIM-1 antibody 3B3, which enhances T cell activation and autoimmunity, RMT1-10 acts as an antagonist, inhibiting pathogenic Th1 and Th17 responses and promoting a Th2 response, offering protection in models like experimental autoimmune encephalomyelitis (EAE).

Summary Table: Key Effects of RMT1-10

Conclusion

RMT1-10 is a TIM-1-blocking monoclonal antibody that modulates B cell (B1a) and T cell responses, leading to increased IgM production and reduced atherosclerosis in mice, as well as prolonged allograft survival and increased immune tolerance in transplant models. Its effects are characterized by a shift from pro-inflammatory Th1/Th17 to anti-inflammatory Th2 responses, often requiring the presence of regulatory T cells for full efficacy. These findings position RMT1-10 as a promising candidate for therapeutic intervention in autoimmune and alloimmune disorders.