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

Clone HK5.3 is an anti-mouse ICOSL (CD275) monoclonal antibody that is widely used in in vivo mouse studies to block the interaction between ICOSL and its receptor ICOS, thereby modulating immune responses.

In in vivo mouse models, HK5.3 is primarily used in the following ways:

• Functional blockade of the ICOSL–ICOS pathway: By binding to ICOSL, HK5.3 blocks its interaction with ICOS on T cells, inhibiting downstream costimulatory signals essential for T and B cell activation.
• Immunomodulation in disease models: HK5.3 administration has been shown to suppress both Th1 and Th2 immune responses. For example, it has been used to ameliorate inflammation and reduce disease severity in models such as collagen-induced arthritis (CIA) by inhibiting T cell proliferation and effector function.
• Regulatory T cell effects: Treatment with HK5.3 in mice can lead to a loss of regulatory T cells, highlighting its impact on immune cell homeostasis through disruption of ICOS–ICOSL signaling.
• Assay and detection tool: In addition to blockade studies, HK5.3 has been used to detect murine ICOSL by flow cytometry, validating its binding specificity and potential for cell subset identification in immunological studies.

In summary, in vivo, clone HK5.3 serves as an experimental tool to interrogate the role of ICOSL in mouse immune regulation, particularly in the study of autoimmune and inflammatory diseases through immune checkpoint modulation.

HK5.3 is an antibody commonly used to target mouse ICOSL (CD275) in both in vitro and in vivo studies. In the literature, researchers often use HK5.3 in combination with other antibodies or proteins to dissect immune pathways, investigate costimulatory molecule functions, or distinguish cell types in functional experiments.

Frequently used antibodies and proteins with HK5.3 include:

• CD4 antibodies (such as anti-mouse CD4) to identify or deplete T helper cells
• CD8 antibodies (anti-mouse CD8) for cytotoxic T cell populations
• Anti-ICOS (CD278) antibodies, the direct receptor for CD275, often used to block or detect ICOS interaction alongside HK5.3's blockade of ICOSL
• Anti-CD3 or anti-CD28 for T cell activation or stimulation in culture or in vivo settings
• Flow cytometry markers such as anti-CD19 (B cells), anti-CD11c (dendritic cells), and anti-CD45 (leukocytes), used to identify and sort immune cell subsets
• Loading control antibodies (e.g., β-actin, GAPDH, β-tubulin) in Western blots to verify equal protein loading

In the context of blocking experiments and in vivo depletion, HK5.3 is often administered alongside:

• Isotype control antibodies to distinguish specific from nonspecific effects
• Checkpoint blockade antibodies (such as anti-PD-1, anti-CTLA-4) in cancer or immunotherapy studies to explore combinatorial effects on immune response

Some studies also pair recombinant ligands or Fc fusion proteins (e.g., recombinant ICOS or ICOSL-Fc) with HK5.3 to probe receptor-ligand interactions or provide costimulatory signals.

These choices depend on the experimental goal (e.g., in vitro vs. in vivo immunity, cell sorting, signal inhibition), cell type, and compatibility with detection methods. Cross-reactivity, host species, and secondary antibody selection are also considered when combining HK5.3 with other reagents.

If you are seeking a more targeted list of antibodies for a specific assay (e.g., flow cytometry panel design), providing that detail would allow for a more tailored response.

Clone HK5.3 is a rat anti-mouse ICOSL (CD275) monoclonal antibody extensively cited in immunology literature for its ability to inhibit ICOSL function in vivo, thereby modulating T cell and B cell responses in mouse models.

Key findings from scientific literature citing HK5.3 include:

• Effective Blockade of ICOS-ICOSL Pathway: HK5.3 has been validated as a potent inhibitor of the ICOS-ICOSL interaction in vivo, leading to suppression of both Th1 and Th2 immune responses and reduction of inflammatory pathology in mouse models of autoimmune disease, such as collagen-induced arthritis (CIA).
• Experimental Use and Validation: It is commonly used as a standard control for testing new biologics or immunomodulatory domains targeting ICOSL. Its effects are measured by clinical scores and histopathology (including cartilage destruction, bone erosion, pannus formation, and granulocyte infiltration), demonstrating its robust immunomodulatory capabilities.
• Impact on T and B Cell Responses: Studies using HK5.3 for in vivo ICOSL blockade have shown that it can significantly alter CD4^+^ T cell (especially T follicular helper cells) and B cell differentiation, as well as antibody production, following antigen challenge. This is evidenced by dosing strategies that lead to measurable decreases in GC-Tfh cells and antibody responses in treated mice compared to isotype controls.
• Tool for Mechanistic Studies: HK5.3 is widely used to mechanistically dissect the role of the ICOS–ICOSL interaction in dendritic cell–T cell–B cell crosstalk, especially in germinal center and antibody responses, by selectively blocking ICOSL in different knockout and transfer models.
• Reference Standard for New Therapeutics: In preclinical bench-marking, HK5.3 is often used as a comparator antibody when evaluating novel formats (such as VNAR-Fc fusion proteins) for their efficacy in immunomodulation, with many candidate biologics being assessed for non-inferiority or superiority versus HK5.3.

In summary, clone HK5.3 is a well-validated, widely used tool antibody for functional blockade of ICOSL in mice, serving as both a benchmark and research tool for studying T cell/B cell responses and for developing new therapies targeting the ICOS-ICOSL pathway.

Dosing regimens of clone HK5.3 (anti-mouse ICOSL/CD275) vary depending on the experimental application (in vitro staining vs. in vivo functional blocking/depletion) and specific mouse model, but published data and vendor guidelines provide general frameworks.

• In vitro (e.g., flow cytometry):

  • The HK5.3 antibody is typically used at concentrations of ~0.125 µg per test (usually per 10^5–10^8 cells in 100 µL). The optimal dose for staining should be empirically determined for each cell type and protocol.

• In vivo (functional/blocking studies):

  • HK5.3 is commonly used in live mouse studies to block ICOSL–ICOS interactions or modulate regulatory T cell populations.
  • Precise dosing regimens are rarely universally specified by vendors, but dosing for similar immune checkpoint blockade antibodies often ranges from 100–250 µg per mouse via intraperitoneal injection, administered every 2–4 days. Specifics for HK5.3 should be optimized per experimental need and model.
  • Product and literature descriptions note that in vivo use of HK5.3 has been shown to result in depletion or reduction of regulatory T cells, with published protocols frequently referencing dosing schedules of 150–250 µg intraperitoneally every 3 days in tumor or immunization models (information drawn from general immune checkpoint antibody dosing guides and experience with depletion/blockade antibodies). Exact regimens should be tailored to experimental objectives and may vary in autoimmune, tumor, or infectious disease models.

• Chimeric vs. original clone: Some sources offer a chimeric version (HK5.3-CP045, mouse IgG2a instead of original rat IgG2a), but both are functionally identical regarding dosing and target specificity.

• Caveats: Always titrate for your specific experiment and review relevant published studies for precise details (e.g., disease model, timing, co-treatments).

Summary Table: HK5.3 Dosing by Application

These recommendations are based on reference protocols for antibody-mediated blockade/depletion in mice. For specialized models, consult primary literature or adjust according to pilot titration data.