Anti-Human CD2 [Clone LO-CD2a] — Purified in vivo PLATINUM™ Functional Grade

In Vivo Applications of Clone LO-CD2a in Mice

Clone LO-CD2a, also known as BTI-322, is a rat monoclonal antibody targeting human CD2—a glycoprotein expressed on T cells, NK cells, most thymocytes, and a small subset of bone marrow cells. While endogenous mouse CD2 is not the primary target of LO-CD2a, the antibody finds significant utility in humanized mouse models—mice engrafted with human immune cells—for several in vivo research applications.

Key In Vivo Applications

• Immunosuppression in Organ Transplant Models: LO-CD2a is primarily used to prevent or treat graft rejection after transplantation by depleting human CD2+ T cells and inhibiting immune responses. It has been shown to effectively prevent or treat acute kidney allograft rejection in humanized mouse models by targeting human T cells that would otherwise drive rejection.
• Depletion of Human CD2+ Immune Cells: Administration of LO-CD2a in humanized mice leads to rapid and sustained depletion of circulating human T cells (and, to a lesser extent, NK cells) through antibody-dependent cell-mediated cytotoxicity (ADCC) mediated by host NK cells and monocytes. This is useful for studying the role of human T cells in disease models or for transiently depleting these cells for mechanistic studies.
• Prevention of Immune Activation and Proliferation: LO-CD2a inhibits the activation and proliferative responses of human T cells in vivo, not only upon initial antigen exposure but also upon subsequent rechallenge, inducing a state of antigen-specific hyporesponsiveness. This mechanism is distinct from some other anti-CD2 antibodies, which may induce transient depletion without long-term hyporesponsiveness.
• Low Cytokine Release Syndrome: In contrast to some other T cell-depleting antibodies (e.g., OKT3), LO-CD2a induces minimal cytokine release, reducing the risk of severe cytokine-mediated side effects in vivo.

Mechanism of Action

• ADCC-Mediated Cell Depletion: The primary in vivo effect is mediated by NK cells and activated monocytes, which recognize the Fc portion of the rat IgG2b antibody bound to human CD2+ cells, leading to targeted cell lysis.
• CD2 Down-Modulation: The antibody induces down-modulation (internalization or shedding) of CD2 from the surface of targeted cells, further inhibiting T cell activation and function.
• Induction of T Cell Hyporesponsiveness: Even after CD2 re-expression, previously exposed T cells remain hyporesponsive to antigen rechallenge, providing durable immunosuppression.

Additional Considerations

• Mouse Limitations: LO-CD2a does not bind murine CD2, so its effects in wild-type mice are negligible unless human immune cells are present. Therefore, most in vivo applications are restricted to humanized mouse models.
• Preclinical Safety: Functional-grade LO-CD2a is rigorously tested for low endotoxin and pathogen content to ensure it does not confound preclinical data in mouse studies.

Summary Table

Conclusion

In vivo, LO-CD2a is chiefly used in humanized mouse models to deplete human CD2+ T cells, prevent graft rejection, and study mechanisms of immunosuppression. Its actions are mediated by ADCC and CD2 down-modulation, leading to durable T cell hyporesponsiveness with minimal cytokine release, making it a valuable tool for transplantation immunology and human immune cell research in mice.

Commonly used antibodies or proteins paired with LO-CD2a in the literature include anti-CD3, anti-TCR Vβ family-specific antibodies, and markers for monocytes and other T cell-related surface proteins.

Key combinations and supporting details:

• Anti-CD3 (clone OKT3): Frequently used alongside LO-CD2a to study T cell activation, depletion, and proliferation, as both target different surface molecules critical for T cell function.
• Anti-TCR Vβ family antibodies: These are used in lineage tracing, T cell subset analysis, and mechanism studies in concert with LO-CD2a, particularly to investigate specificity and depletion of T cell receptor subtypes.
• Monocyte and surface interaction markers: Research often includes monocyte markers or related immune cell markers to explore the role of monocyte interaction (e.g., Fc-mediated depletion mechanisms) when using LO-CD2a.
• Other anti-CD2 clones: Comparative studies may use additional anti-CD2 monoclonals such as Leu-5b, OKT11, and MT910 to differentiate the effects or mechanisms of LO-CD2a.
• Ligand/partner proteins: Proteins like CD58 (LFA-3), CD48, CD59, and CD15 are important in the biology of CD2 and are referenced in mechanistic work involving LO-CD2a for both structural and interaction studies.
• Isotype controls: Rat IgG2b-κ isotype controls are commonly run in parallel as experimental controls.

These combinations reflect common research aims such as T cell depletion, tolerance induction, immunosuppression, and dissecting specific immune cell interactions in transplantation or autoimmunity models.

The key findings from clone LO-CD2a citations in scientific literature center on its unique immunosuppressive mechanisms, effective T cell depletion, and induction of antigen-specific immune unresponsiveness, distinguishing it from other anti-CD2 antibodies.

Main findings include:

• Immunosuppressive activity: LO-CD2a potently inhibits T cell activation and proliferation in vitro, including strong suppression of mixed lymphocyte cultures (MLC/MLR) and peripheral blood mononuclear cell (PBMC) proliferation stimulated by agents like OKT3.
• T cell depletion in vivo: Administration causes efficient depletion of CD2+ T cells through mechanisms such as antibody-dependent cell-mediated cytotoxicity (ADCC), primarily via natural killer (NK) cells and monocytes.
• Low cytokine induction: Unlike some other anti-CD2 clones, LO-CD2a induces very low levels of cytokine release and inhibits cytokine production, limiting potential toxicities associated with cytokine storm.
• Induction of hyporesponsiveness: After initial exposure, T cells become specifically unresponsive to restimulation with the same antigen but retain responses to third-party antigens, demonstrating a form of induced "tolerance." This distinguishes LO-CD2a from other anti-CD2 antibodies.
• Role in transplantation: Developed for use in organ allotransplantation, LO-CD2a helps prevent graft rejection by depleting effector T cells and interfering with both primary and memory alloantigen responses.
• Mechanism specificity: The induction of tolerance/unresponsiveness requires interaction between the antibody's Fc portion and monocytes, and involves activation-associated T cell depletion.
• CD2 expression details: The target antigen, CD2, is broadly expressed on mature human T cells, most thymocytes, NK cells, and a minority of bone marrow cells, making LO-CD2a effective for depleting these populations.

These findings collectively position LO-CD2a as a distinctive tool for immunosuppression and tolerance induction, with applications in transplantation and preclinical immune modulation studies.

Dosing regimens of clone LO-CD2a (anti-human CD2 monoclonal antibody) vary across mouse models and are not standardized; they are typically determined by the experimental context, especially the presence of human T cells in humanized or chimeric mice, since LO-CD2a targets human CD2 and does not react to mouse CD2.

• Humanized Mouse Models: In studies using humanized mice (hu-SCID or chimeric models with human T cells), LO-CD2a is dosed based on the goals of the experiment, such as T cell depletion or graft rejection inhibition. Here, dosing generally follows protocols similar to those for other human-targeted antibodies, often using doses ranging from low microgram quantities for efficacy and safety assessments, but the specific dose must be determined empirically for each study.

• MLR (Mixed Lymphocyte Reaction) Assays: In in vitro and ex vivo lymphocyte proliferation models (MLR), LO-CD2a (also termed BTI-322) is often added at 100–200 ng/ml in cell culture assays to inhibit T cell responses or proliferation. For example, 200 ng/ml LO-CD2a was used for MLR inhibition in vitro; timing of addition (Day 0, 1, or 2) can affect the inhibitory outcome.

• In Vivo Studies (Transplant/Immunosuppression): Although in vivo dosing in mice is less frequently reported and variable, doses are typically extrapolated from comparable monoclonal antibodies:

  • For similar human-targeted antibodies (e.g., OKT3, Campath-1H), researchers used 2–10 µg per mouse via intraperitoneal (IP) or intravenous (IV) injection, adjusting based on toxicity and efficacy. For ATG (another immunosuppressive antibody), doses ranged from 30–150 µg per mouse.
  • Severity of effects with such agents increases with dose and choice of administration route: IV dosing leads to stronger, more rapid effects compared to IP.
  • LO-CD2a protocols in transplantation or immunosuppressive studies are normally modeled after these agents but require dose titration due to potential differences in toxicity and efficacy in different mouse models.

• Experimental Context and Non-Standardization: No universal dosing regimen exists. Variables influencing dosing include:

  • Mouse background (immunodeficient, humanized, transgenic expression of human CD2)
  • Immune cell composition (need for human T cells)
  • Route of administration (IP vs. IV)
  • Intended immunological outcome (depletion vs. inhibition).

• Summary Table:
  | Mouse Model Type | Typical Dose Range | Route | Notes ||----------------------------|---------------------------|---------------|----------------------------------------------------|| Humanized (hu-SCID) | 2–10 µg/mouse | IP, IV | Dose, route, timing empirically determined || In vitro/ex vivo MLR | 100–200 ng/ml (culture) | n/a | For T cell proliferation inhibition || Other monoclonals (ref.) | 2–150 µg/mouse | IP, IV | Used for reference, actual LO-CD2a dose varies |

Protocols should be customized for each experimental setup, with close consideration of mouse strain, engraftment of human cells, and desired immunological effect.

If further precision is needed, dose titration and pilot studies are advised before definitive experiments, due to possible severe reactions at higher doses, particularly with IV administration.