Anti-Mouse ICOSL (CD275) (Clone HK5.3) – Purified in vivo PLATINUM™ Functional Grade

Clone HK5.3 is a monoclonal antibody targeting mouse ICOSL (CD275) and is commonly used in vivo in mice to block the interaction between ICOSL and its receptor ICOS, thereby inhibiting ICOS-mediated co-stimulatory signaling in the immune system.

Common in vivo applications in mice include:

• Functional blockade of ICOSL: HK5.3 is routinely used to block the ICOSL-ICOS interaction in vivo, thereby inhibiting co-stimulatory signaling that is critical for T cell and B cell activation and function.
• Immunomodulation studies: Treatment with HK5.3 has been shown to result in altered immune cell populations, such as the reported reduction of regulatory T cells (Tregs) in mice.
• Autoimmunity and inflammation models: The antibody has been used in murine models of autoimmune diseases (e.g., collagen-induced arthritis), where it inhibits both Th1 and Th2 immune responses and ameliorates disease by suppressing T cell help.
• Infection and tumor models: HK5.3 is also used to evaluate the impact of ICOSL-mediated co-stimulation on T cell responses in viral infections and cancer immunology, helping to elucidate the role of ICOS signaling in immunity during these conditions.
• Mechanistic studies: Blocking ICOSL with HK5.3 enables detailed assessment of ICOS pathway involvement in immune regulation, tolerance, or breakdown thereof.

In these in vivo settings, HK5.3 is typically administered via intraperitoneal injection or other systemic routes to achieve neutralization/blockade of ICOSL on antigen-presenting cells (e.g., B cells, dendritic cells, macrophages), thereby allowing researchers to dissect the importance of ICOS:ICOSL signaling in various immunological processes.

Key details:

• Application types: Blocking/neutralization, mechanistic studies, disease modeling (autoimmunity/infection), and regulatory T cell research.
• Validated protocols: Injections (intraperitoneal or intravenous), with dosing and schedules varying by application and study design.

No substantial evidence was found for other uses outside these immunological and blocking applications in mice. HK5.3 is widely reported as an in vivo research tool to study the function of ICOSL in mouse models of immunity and disease.

Commonly used antibodies and proteins alongside HK5.3 (anti-mouse ICOSL/CD275) in the literature depend on the experimental context. However, studies using HK5.3 generally involve:

• ICOS (CD278) antibodies: Since HK5.3 targets ICOSL (the ligand), antibodies to the receptor ICOS are frequently used in combination to investigate ICOS/ICOSL-mediated signaling pathways or to block/reconstitute the ligand-receptor interaction when analyzing T cell costimulation or immune checkpoint roles.
• Other B7 family proteins: Antibodies against related costimulatory molecules, such as CD80 (B7-1), CD86 (B7-2), and PD-L1 (B7-H1), are often employed for comparative studies or to control for effects specific to ICOSL inhibition.
• Immune cell surface markers: Antibodies to markers including CD4, CD8, CD19, CD11c, F4/80, and others are routinely used to phenotype immune subsets in tissues when measuring HK5.3-mediated changes in T/B cell responses or antigen-presenting cell status.
• Regulatory T cell markers: Since ICOSL/ICOS is important for regulatory T cell homeostasis, studies may use antibodies to detect and quantify Foxp3 and CD25 in conjunction with HK5.3.
• Functional readouts and controls: Isotype controls, and antibodies detecting activation markers (CD69, CD25) or proliferation (e.g., Ki67, BrdU), are standard in functional analyses alongside HK5.3.

Protein partners or related reagents include:

• Mouse ICOS-Fc fusion proteins: Used for binding/competition assays with HK5.3 to confirm blocking activity.
• Recombinant ICOSL-Fc or B7-H2 fusion proteins: Used in ELISA or flow cytometry to assay HK5.3 specificity or affinity.
• Variants of HK5.3 (e.g., LALA-PG Fc mutants): These engineered versions (with reduced effector function) are used in experiments where FcγR engagement is undesirable.

Most co-usage combinations are detailed in immunophenotyping, immune checkpoint, and functional blocking assay publications using the above tools along with HK5.3.

If you require a list of specific clone names or additional technical details for standard pairs or experimental controls used with HK5.3, please clarify the research context (e.g., in vivo depletion, flow cytometry, or checkpoint blockade models).

The key findings from scientific literature using the HK5.3 clone—a monoclonal antibody specific for mouse ICOSL (CD275/B7-H2)—center on its roles in immunology research, especially regarding T cell costimulation, regulatory T cell homeostasis, and functional blockade studies.

Major findings and applications of clone HK5.3:

• Functional Blockade of ICOS/ICOSL Pathway: HK5.3 is widely used to block the interaction between ICOSL (on antigen-presenting cells) and ICOS (on activated T cells), disrupting an important co-stimulatory signal for T cell proliferation, cytokine secretion, and B cell responses. This blockade is used in both in vitro and in vivo experiments for mechanistic studies.

• Impact on Regulatory T Cells (Tregs): Treatment of mice with HK5.3 has been shown to lead to loss of regulatory T cells, implicating the ICOS-ICOSL axis in the maintenance of Treg homeostasis. For example, HK5.3-based blockade reduced Treg expansion in models of acute myeloid leukemia, affecting immune regulation.

• Use in Autoimmunity and Inflammation Studies: HK5.3-mediated ICOSL blockade is utilized to delineate the role of ICOSL in autoimmune disease models, such as rheumatoid arthritis, and to analyze how ICOSL-expressing B cells regulate autoimmune inflammation. Its application helps clarify the function of co-stimulatory pathways in disease progression.

• Identification and Quantification via Flow Cytometry: HK5.3 is a validated tool for flow cytometric analysis, staining mouse splenocytes and other tissues to quantify ICOSL expression patterns.

• Dendritic Cell and Macrophage Research: HK5.3 has been used to explore how ICOSL expression on dendritic cells and macrophages controls their maturation and function—in turn regulating adaptive immune responses.

• Broad Research Applications: HK5.3 is cited in studies investigating immune modulation during viral infections (e.g., cytomegalovirus), vaccine responses, and nanoparticle-mediated immune manipulation.

Summary Table: Key Research Uses of HK5.3

Caveats: Citations found span in vitro and in vivo applications, and effects may depend on model and context (disease state, immune background, etc.). The antibody's functional performance (e.g., required concentration) can vary and is often titrated for each model.

Overall, HK5.3 is a well-established research antibody for dissecting the role of ICOSL in mouse immunology, particularly in T cell biology, regulatory T cell maintenance, and immune regulation in health and disease.

The dosing regimens for clone HK5.3, an anti-mouse ICOSL (CD275) antibody, vary considerably across different experimental contexts and research objectives in mouse models.

Standard Dosing Parameters

The HK5.3 antibody is typically administered via intraperitoneal injection in mouse models. This rat IgG2a monoclonal antibody blocks the binding of ICOSL to ICOS and has been shown to lead to a loss of regulatory T cells in treated mice.

Context-Specific Dosing Regimens

Hydrodynamic Tail Vein (HDTV) Injection Models

In studies using HDTV-injected wild-type C57Bl/6 mice, researchers employed a dose of 100 μg per mouse administered intraperitoneally on days 0, 3, 6, and 9 after HDTV injection. This schedule represents a relatively frequent dosing approach with treatment occurring every 2-3 days during the acute experimental phase.

Atherosclerosis Models

For long-term treatment in apolipoprotein E-deficient mice fed a western diet, a different strategy was used. Mice received α-ICOSL antibody twice a week for 6 weeks. This represents a more sustained, lower-frequency dosing regimen appropriate for chronic disease models where prolonged ICOSL blockade is desired.

ADAM10-Mediated Shedding Studies

When investigating ICOSL shedding on B cells, researchers utilized a sub-optimal dose of 10 μg per mouse administered every other day. This notably lower dose compared to the 100 μg standard was deliberately chosen to reduce exposure levels while maintaining sufficient biological activity to observe experimental effects.

Comparison to Related Antibodies

The dosing of HK5.3 differs from other immune checkpoint and depletion antibodies commonly used in mouse models. For instance, anti-CTLA-4 antibodies typically use 100-250 μg per mouse every 3 days, while CD4 or CD8 depletion antibodies require 200-250 μg per mouse administered 2-3 times per week. The variability in HK5.3 dosing—from 10 μg to 100 μg—reflects the different biological endpoints being measured, whether complete ICOSL blockade or partial modulation of the ICOS-ICOSL pathway is required.