Anti-Mouse CD28 [Clone D665] — Purified in vivo PLATINUM™ Functional Grade

Common In Vivo Applications of Clone D665 in Mice

Clone D665 is a monoclonal antibody targeting mouse CD28, a key costimulatory receptor expressed on thymocytes, most peripheral T cells, and NK cells. Its primary use in vivo is as a CD28 superagonist, capable of robustly activating CD28 signaling and inducing T cell proliferation even in the absence of antigen-specific T cell receptor (TCR) stimulation.

Key Applications

• Induction and Expansion of Regulatory T Cells (Tregs): D665 is most commonly used to expand immunosuppressive regulatory T cells (Tregs) in vivo in various mouse models of disease. This application exploits the unique property of CD28 superagonists to preferentially activate and expand Treg populations, which can then modulate immune responses or suppress inflammation.
• Prevention of Inflammation and Autoimmunity: By expanding Tregs, D665 has been shown to prevent inflammation and autoimmune responses in experimental settings. For example, in vivo treatment of mice with D665 resulted in rapid expansion and activation of Tregs, which prevented cytokine storm and dampened inflammatory responses.
• Generation of Type 1 Regulatory T Cells (Tr1): D665 has been used in combination with other immunomodulatory antibodies (e.g., anti-GITR mAb G3c) to induce IL-10/IFN-γ–co-producing Tr1 cells in vivo. This combination can lead to permanent allograft acceptance in a Tr1-dependent manner, highlighting its therapeutic potential in transplantation.
• T Cell Activation and Proliferation Studies: Beyond Tregs, D665 is employed to study general T cell activation and proliferation in vivo, independent of TCR engagement, making it a valuable tool for dissecting the roles of costimulatory signals in immune responses.

Standard Experimental Protocols

• Dose and Route: A common protocol involves a single intraperitoneal (i.p.) injection of D665, for example, 100 μg/mouse in 100 μL PBS. The timing of administration varies depending on the experimental design.
• Disease Models: D665 has been applied in various mouse models of autoimmune, inflammatory, and transplantation diseases to study immune regulation and tolerance.

Summary Table

Conclusion

Clone D665 is a powerful tool in mouse immunology research, primarily used to expand Tregs and modulate immune responses in vivo, with applications ranging from basic research on T cell activation to preclinical studies in autoimmunity and transplantation. Its ability to act as a CD28 superagonist makes it uniquely suited for studies requiring robust, antigen-independent T cell stimulation.

The most commonly used antibodies or proteins with D665 (a CD28 superagonist antibody) in the literature are:

• Anti-GITR antibodies (notably G3c and DTA-1): These are frequently used in combination with D665 to study regulatory T cell (Treg) and type 1 regulatory T cell (Tr1) induction and function. In particular, G3c is used sequentially after D665 to enhance Tr1 cell generation and induce immunological tolerance, such as in transplantation models.
• DTA-1, another anti-GITR antibody, has been used for comparative and mechanistic studies alongside D665 to investigate different outcomes in Treg and effector T cells.

Additional proteins and markers studied in combination with D665, particularly in immunological contexts, include:

• Foxp3: Used as a marker for Treg cells, whose expansion is a primary effect of D665.
• Cytokines such as IL-10 and IFN-γ: D665 in combination with G3c leads to the generation of IL-10/IFN-γ-co-producing Tr1 cells.
• Transcription factors Prdm1 (Blimp-1) and Maf: Studied for their roles in the D665-G3c-induced Tr1 differentiation pathway.
• MAPK-STAT3 signaling pathway proteins: Investigated in the mechanistic analysis of how G3c enhances the effect of D665.

Experimental contexts and other combinations:

• The use of D665 with other standard immunological reagents such as anti-CD3 (for T cell activation in vitro), and with markers for T cell phenotyping and functional analysis is implied but less explicitly detailed in the key studies.

Summary Table: Major antibodies/proteins used with D665

Key point: The literature most often describes D665 used with anti-GITR antibodies (especially G3c), plus standard T cell markers and cytokines to assess immune modulating effects and mechanisms.

Clone D665 is cited in scientific literature as a mouse anti-CD28 monoclonal antibody that functions as a CD28 superagonist, with its primary use being the in vivo expansion of regulatory T cells (Tregs) and investigation of immunoregulatory mechanisms in various murine disease models.

Key findings from D665 citations include:

• Distinct expansion of Treg cells: D665 has been shown to preferentially expand Treg cells (regulatory T cells) over effector T cells (Teff) in multiple rodent models, contributing to Treg-mediated dampening of autoimmune and inflammatory responses.
• Prolonged allograft survival, but not permanent acceptance: In mouse cardiac allograft models with strong rejection responses, D665 treatment led to prolonged graft survival but stopped short of inducing permanent graft acceptance.
• Costimulatory activity independent of TCR ligation: D665 binds to the CD loop of CD28 and induces T cell proliferation without requiring TCR (T cell receptor) engagement, which classifies it as a superagonist.
• Gene expression shifts and Tr1 cell induction: Combination treatments involving D665 (notably with anti-GITR antibodies) result in substantial generation of Type 1 regulatory (Tr1) cells, reflected in transcriptomic studies showing thousands of differentially expressed genes in T cells after treatment (with IL-10 being highly upregulated).
• Mechanistic insight for immune modulation: D665-mediated costimulation upregulates GITR expression on both Treg and Teff cells, setting the stage for subsequent modulation by additional immunotherapies (e.g., anti-GITR antibodies) to further expand or differentiate regulatory populations.

Additional details:

• D665 is used extensively in mouse immunology research, with commercial suppliers providing the antibody for both in vitro and in vivo assays.
• The antibody is unconjugated and purified, engineered for experiments requiring consistent expansion/activation of T cells, especially for regulatory or tolerogenic studies.

Overall, clone D665 is a well-established immunological tool for modulating T cell responses, particularly to expand regulatory T cells and study tolerance, immune suppression, and allograft survival mechanisms in preclinical mouse models.

Dosing Regimens of Clone D665 Across Mouse Models

Clone D665 is an anti-mouse CD28 monoclonal antibody widely recognized as a CD28 superagonist, utilized primarily to induce the expansion of regulatory T cells (Tregs) in various mouse models of disease. However, despite its established biological role, detailed and systematic data on D665 dosing regimens across different mouse models are not explicitly reported in the available literature or these search results.

What Is Known

• Common Use: Clone D665 is most commonly used to expand Treg populations in vivo, but the specific dosing (amount, frequency, route) is not detailed in the cited sources.
• Example Application: In a mouse heart transplantation model, D665 was used in combination with another antibody (G3c) to induce permanent allograft acceptance, but again, the exact D665 dose is not specified in the search results.
• General Antibody Dosing in Mice: Other anti-mouse antibodies (e.g., anti-CTLA-4, anti-CD4, anti-CD8) are typically administered at 100–250 µg per mouse, intraperitoneally, every 2–3 days, but these are not D665 and should not be assumed equivalent without evidence.
• No Publicly Available Protocol: From other cited sources provide a standardized dosing table or protocol for D665, unlike for other antibodies where dosing is well documented.

Gaps and Limitations

• Model-Specific Variability: Mouse models differ widely in purpose (e.g., autoimmunity, transplantation, cancer), and optimal dosing may depend on the specific experimental context (disease severity, desired Treg expansion, toxicity limits).
• No Direct Citations: The recent literature and antibody supplier documentation do not specify if D665 dosing varies by model, nor provide a comparative table of doses used in different settings.
• Need for Primary Literature: For precise, model-specific D665 dosing, researchers typically refer to original peer-reviewed studies or contact the antibody supplier for unpublished data.

Practical Recommendations

Given the lack of explicit data in these results, if you are planning to use clone D665 in a mouse model, you should:

• Consult Original Research Papers using D665 in your model of interest for exact dosing details.
• Consider General Principles: Start with doses in line with other in vivo mouse antibodies (e.g., 100–250 µg per mouse, intraperitoneal, every 2–3 days), but validate empirically for your model and endpoint.
• Optimize Empirically: Given potential model-specific differences, pilot studies to assess efficacy and toxicity are crucial.

Summary Table: Available Data on D665 vs. Other Mouse Antibodies

Table shows that for D665, specific dosing details are missing, unlike other commonly used antibodies.

Conclusion

There is no published, systematic comparison of D665 dosing regimens across different mouse models in the available literature. While D665 is established as a CD28 superagonist for Treg expansion in mice, researchers must rely on individual study protocols, supplier recommendations, and empirical optimization for their specific model. Always consult primary literature and antibody suppliers for the most accurate and model-appropriate dosing guidance.