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

Clone D665 is most commonly used in vivo in mice to induce the expansion and activation of regulatory T cells (Tregs) as well as other immunomodulatory T cell subsets via superagonistic stimulation of the CD28 receptor.

Key applications include:

• Treg Expansion and Activation: D665 is prominently used to activate Tregs, resulting in their rapid expansion in mouse models. This is a central tool for studying immune regulation and tolerance in contexts such as autoimmune disease, transplantation, and inflammation.
• T Cell Stimulation (Antigen-Independent): As a CD28 superagonist, D665 can stimulate T cell proliferation in vivo without the need for concurrent TCR (antigen) engagement, making it valuable for dissecting costimulatory signaling pathways.
• Induction of Tr1 Cells (Tolerogenic T Cells): D665, especially in combination with anti-GITR mAb (G3c), has been used to induce Tr1 cells (IL-10/IFN-γ–co-producing, Foxp3− regulatory cells) in vivo, for the purpose of establishing transplantation tolerance and suppressing unwanted immune responses.
• Prevention of Cytokine Storm: The rapid expansion of Tregs triggered by D665 has been shown to prevent aberrant cytokine production (cytokine storm) in certain disease models.

Additional context:

• Disease Models: D665 is routinely applied in mouse models of autoimmune diseases, transplantation, and immunopathology where controlled immune suppression or tolerance is desired.
• Mechanism: The antibody acts as a superagonist for CD28, meaning it potently triggers CD28-mediated signaling pathways, leading to preferential activation/expansion of immunosuppressive T cell subsets rather than proinflammatory T cell responses in mice.

In summary, the predominant in vivo research applications of clone D665 in mice are centered on expanding and activating regulatory T cells to study immune tolerance, suppression, and transplantation outcomes, as well as on driving T cell proliferation independent of antigen stimulation for mechanistic immunology research.

Commonly used antibodies or proteins in the literature with D665 (an anti-mouse CD28 superagonist) include:

• Anti-GITR antibodies: The most prominent combination is with G3c, an anti-GITR monoclonal antibody, used to induce regulatory type 1 (Tr1) cells in vivo for transplantation tolerance studies. Studies also mention DTA-1, another anti-GITR antibody, which is used in immune modulation research, particularly in tumor models.
• Foxp3 detection antibodies: Since D665 is known to expand regulatory T cells (Tregs), flow cytometry panels often include antibodies against Foxp3, the master transcription factor of Tregs, to quantify or characterize these populations.
• Lineage antibodies: Standard flow cytometry panels typically include antibodies against CD4 and CD8 to distinguish T cell subsets after D665 stimulation, as well as other markers such as CD25 (IL-2Rα) and CD44 for activation states.
• Cytokine detection reagents: Antibodies against IL-10, IFN-γ, and sometimes IL-2 are used in intracellular staining to assess cytokine profiles of cells expanded or activated by D665.
• Markers of signaling or differentiation: Studies investigating the molecular mechanisms might use antibodies against transcription factors or signaling proteins such as Prdm1 (Blimp-1), Maf, or phosphorylated forms of signaling molecules in pathways like MAPK and STAT3.
• Control antibodies: Isotype controls such as purified mouse IgG1 or rabbit IgG (depending on experiment design) are commonly included as negative controls to ensure specificity.

Table of commonly used antibodies/proteins with D665:

Most recent and translational studies use D665 in conjunction with G3c anti-GITR mAb for robust Tr1-mediated tolerance, supporting their combined application in immune modulation models. Additional lineage, activation, and cytokine markers expand the characterization of cellular responses to D665 in experimental settings.

Clone D665 is a monoclonal antibody targeting mouse CD28 that has emerged as an important research tool, with key findings demonstrating its unique properties as a CD28 superagonist and its therapeutic potential in immune modulation.

Mechanism of Action and Immunological Properties

D665 functions as a CD28 superagonist that binds to the laterally exposed CD loop of CD28 and induces T cell proliferation without requiring T cell receptor (TCR) ligation. This distinguishes it from conventional CD28 agonists that typically require concurrent TCR engagement. The antibody reacts with mouse CD28, a 45 kDa costimulatory receptor expressed by thymocytes, most peripheral T cells, and NK cells.

Regulatory T Cell Expansion

The most prominent finding across multiple studies is D665's preferential ability to expand regulatory T cells (Tregs) over effector T cells in various rodent models of autoimmune and inflammatory disease. This selective expansion makes it particularly valuable for Treg-based therapeutic interventions. When administered in vivo, D665 induces robust Treg cell expansion, which has been consistently observed across different experimental models.

Generation of Type 1 Regulatory T Cells

A particularly innovative finding involves the sequential combination therapy using D665 followed by anti-GITR antibody treatment. Research demonstrated that D665 treatment strongly upregulates GITR expression on both Treg and effector T cells. When followed by administration of G3c (an agonist anti-GITR antibody), this sequential approach generated large amounts of Type 1 regulatory (Tr1) cells, a specialized subset of IL-10-producing regulatory T cells.

Transcriptomic analysis of these induced Tr1 cells revealed 1,963 differentially expressed genes compared to conventional CD4+ T cells, with IL-10 being the most upregulated gene. This mechanism represents a novel approach to inducing immunosuppressive cell populations with potential applications in transplantation and autoimmune diseases.

Therapeutic Applications in Transplantation

In cardiac allograft models using the BALB/c to B6/J strain combination, D665 treatment alone resulted in prolonged cardiac allograft survival rather than permanent acceptance. This suggests that while D665 monotherapy provides significant immunomodulation, combination approaches may be necessary for achieving long-term tolerance in strong rejection scenarios.

Research Applications

D665 has been utilized in multiple experimental contexts, including in vitro and in vivo T cell stimulation and activation studies. The antibody is available as a research-grade reagent with high purity (≥95%) and low endotoxin levels (≤1EU/mg), making it suitable for controlled experimental work.

Dosing regimens of clone D665 (an anti-mouse CD28 superagonist antibody) vary across mouse models, but published details on specific regimens per disease context are limited. Most sources emphasize its common use to expand regulatory T cells (Tregs) in vivo, but do not provide exact dosage schedules across diverse models.

Essential context and supporting details:

• Common dosing rationale: D665 is primarily employed to induce Treg expansion, which underlies its application in models of transplantation, autoimmunity, and inflammation.

• Route of administration: Standard practice for in vivo monoclonal antibody administration in mice is intraperitoneal (IP) injection, and this is expected for D665 as well, though the specific route is not always stated in available summaries.

• Application in transplantation: In a mouse heart transplantation model, D665 was used in combination protocols to induce permanent allograft acceptance. However, the exact dosing schedule in this reference is not specified; only the positive therapeutic effect is described.

• Commercial protocols: Bio X Cell identifies clone D665 as being "most commonly used to induce expansion of Treg cells in vivo in various mouse models," but their public documentation does not list a fixed mg/kg dose or schedule.

• Comparison to typical antibody dosing: For reference, other immunomodulatory antibodies in mice are administered at doses typically ranging from 100–250 μg per mouse, given intraperitoneally every 3–7 days, depending on the biological endpoint and antibody type.

Limitations and inferences:

• No current search result provides a table or systematic comparison of D665 regimens in different mouse models.
• Available data suggest that, like other immune-targeting antibodies, D665 would be dosed at 100–250 μg per mouse intraperitoneally, every 3–7 days, but this is inferred from general antibody dosing guidelines and must be confirmed by consulting specific publications or manufacturer datasheets per model.

Summary of key findings:

• Dosing of D665 varies depending on the disease model, with most sources highlighting its use for Treg expansion and in transplantation models; however, definitive, model-specific regimens are not standardized in the public domain.
• If exact regimen details are required for a specific disease or study, primary literature or direct manufacturer protocol consultation is necessary, as dosing is likely adjusted based on the desired level of Treg expansion, disease model, and experimental endpoint.