Anti-Rat CD28 (Clone JJ319) – Purified in vivo GOLD™ Functional Grade

Based on the available information, clone JJ319 is actually not used in mice but rather specifically targets rat CD28. This is an important distinction, as JJ319 is a mouse monoclonal antibody developed against rat CD28 and shows reactivity only with rat species.

Primary Applications in Rats

The JJ319 antibody is primarily used for in vitro rather than in vivo applications in rats. The main functional applications include:

T Cell Costimulation Studies: The antibody is used to augment T cell proliferation and enhance IL-2 production in vitro. Soluble JJ319 costimulates the proliferative responses of both CD4+ and CD8+ T cells when combined with anti-αβ-TCR antibodies.

Flow Cytometry: JJ319 is commonly employed for flow cytometric analysis of rat splenocytes and other lymphoid tissues to identify and characterize CD28-expressing T cells.

Immunoprecipitation: The antibody can be used to isolate CD28 protein complexes from rat cell lysates for downstream analysis.

Limited In Vivo Evidence

While the search results indicate that JJ319 is available in formats suitable for in vivo use (such as low endotoxin, functional grade preparations), there is minimal documentation of actual in vivo applications. One reference mentions that an alternate anti-rat CD28 antibody (clone JJ316) is capable of directly stimulating T cells in vivo, but similar in vivo data for JJ319 itself is not provided in the available sources.

Mouse CD28 Antibodies

For in vivo studies in mice, researchers would need to use different antibodies specifically targeting mouse CD28, such as clone PV-1, rather than the rat-specific JJ319 clone.

The antibody JJ319 is most commonly used for detection and modulation of rat CD28, a costimulatory molecule on T cells. In the literature, JJ319 is frequently combined with the following antibodies and proteins:

• R73 anti-rat TCR (T-cell receptor): This antibody is used alongside JJ319 to activate T cells (R73 provides the primary signal via TCR, and JJ319 provides the costimulatory signal via CD28).
• MR1 (anti-rat CD154/CD40L): Often co-administered in transplantation and tolerance studies to modulate costimulatory signals.
• Anti-CD3 antibodies: Used for broad T cell activation, often in conjunction with costimulatory or inhibitory signals involving CD28.
• Anti-rat CD25 (IL-2 receptor α chain): For identifying or depleting regulatory T cells, especially when looking at tolerance or immunosuppression.
• Foxp3 (regulatory T cell marker): Used in flow cytometry or histology to assess the impact of JJ319 treatment on regulatory T cells.
• Anti-CD45: A pan-leukocyte marker often used for gating immune cell populations when analyzing effects of JJ319 in flow cytometry panels.
• CD80 and CD86: As proteins/antigens, these are the natural ligands of CD28; their expression or blockade is often monitored or manipulated in JJ319 studies.

Additionally, general panels for rat immunology studies using JJ319 may also include:

• CD4 and CD8: For delineating helper and cytotoxic T cell subsets.
• CD161: For NK and some T cell subset analyses.
• CD68: For monocyte/macrophage population studies.

In mechanistic and signaling studies, antibodies used with JJ319 have included:

• Anti-phosphotyrosine (e.g., clone 4G10): For detecting signaling via immunoblot.
• Anti-ZAP-70 and nuclear factor antibodies (NF-κB, c-rel, USF-2): To study downstream effects of CD28, especially in research dissecting signaling pathways.

Summary Table: Common Antibodies/Proteins Used with JJ319

These combinations reflect standard practices in rat immunology, transplantation, and T cell activation studies using JJ319.

Key Findings from clone JJ319 Scientific Literature

JJ319 is a mouse monoclonal antibody (mAb) that specifically recognizes rat CD28, a co-stimulatory receptor critical for T cell activation. The scientific literature primarily uses JJ319 for comparative studies on CD28 signaling and to distinguish the effects of conventional versus superagonistic CD28 antibodies.

Functional and Binding Specificity

• JJ319 is a conventional anti-CD28 monoclonal antibody: It does not stimulate robust T cell activation in vivo, unlike other "superagonist" antibodies (e.g., JJ316). For example, administration of JJ319 (0.1 mg) in animal models did not significantly alter the clinical course of experimental autoimmune encephalomyelitis (EAE), suggesting it lacks potent agonist activity.
• Epitope Mapping: The epitope recognized by JJ319 on rat CD28 is located between amino acids 66 and the transmembrane domain. Specifically, a valine residue at position 98 in rat CD28 is critical for JJ319 binding. Mutation of this residue disrupts JJ319 binding, demonstrating the importance of this region for antibody specificity. In contrast, the superagonist JJ316 binds to a different region (aa 37–66), known as the C′′D loop.
• Proximity to the B7 Binding Site: The site bound by JJ319 (near aa 98) is close to the B7 binding domain, which is the natural ligand for CD28. This proximity is relevant for understanding how antibody binding might influence CD28's natural ligand interactions.

Comparative Effects

• Lack of Agonist Activity: While JJ316 (a superagonist) can expand regulatory T cells and confer protection from autoimmune disease, JJ319 at comparable doses does not produce these effects, highlighting functional differences between conventional and superagonistic CD28 antibodies.
• Immunophenotypic Staining and Controls: In experimental studies, JJ319 is commonly used as a control mAb to separate the effects mediated by conventional CD28 engagement from those induced by superagonistic antibodies.

Applications in Research

• Phenotypic Analysis: JJ319 is routinely used for staining T cells in flow cytometry and other assays to confirm CD28 expression.
• Neutralizing and Functional Assays: While JJ319 binds to CD28, it generally does not block the binding of the natural ligand (B7), nor does it induce strong cellular responses, making it a useful tool for defining distinct modes of CD28 engagement.

Summary Table: JJ319 Properties

Conclusion

Clone JJ319 is a conventional antibody to rat CD28, primarily used as a research tool for flow cytometry, immunohistochemistry, and as a negative control in functional studies of CD28 signaling. Its binding is specific to the extracellular region near the B7 binding site, and it does not induce strong T cell activation, which distinguishes it from superagonistic CD28 antibodies like JJ316. These distinctions are critical for understanding CD28 biology and for designing experiments that dissect different aspects of CD28-mediated T cell regulation.

Dosing regimens of clone JJ319 (an anti-rat CD28 monoclonal antibody) vary across mouse models primarily by experimental purpose, dosing frequency, and total dose per injection. However, JJ319 is specific for rat CD28 and thus is predominantly used in rat models or in mice engrafted with rat cells expressing CD28, not typical wild-type mouse models.

Key points based on available data:

• Mouse Strain: In vivo studies with JJ319 generally involve specialized models (e.g., mouse models with rat immune cells, or xenotransplantation setups), since standard mouse CD28 is not recognized by JJ319.
• Dose Range: Published regimens administer typical JJ319 doses in the range of 20 μg to 100 μg per animal, delivered every other day for up to 8 days or as a single 100 μg dose, with similar effects observed between single and repeated dosing.
• Route of Administration: Doses are commonly delivered via intraperitoneal injection, but the precise route may depend on the specific model.
• Study Design Variability:
  • In some GvHD (graft-versus-host disease) models, mice received 100 μg intraperitoneally, every other day or as a single dose.
  • In experiments comparing dosing schedules, both regimens (single vs. repeated) produced comparable results in terms of survival and immune modulation.

Table: Example JJ319 Dosing Regimens in Mouse Models with Rat CD28

Important considerations:

• JJ319 is not used in standard mouse models due to lack of cross-reactivity with mouse CD28.
• Dosing may be tailored to experimental needs such as depletion of specific T cell subsets, immune modulation, or tolerance induction, and pilot experiments are often required.
• Compared to other in vivo antibodies (e.g., anti-PD-1, anti-CTLA-4 for mouse targets), the JJ319 dosing falls in a similar μg range, but is strictly for models involving rat CD28.

If using other anti-CD28 antibodies or anti-mouse CD28 clones in standard mouse models, regimens will differ substantially; the information above pertains only to JJ319 and relevant chimeric or engraftment settings.

In summary, JJ319 dosing regimens in mouse models vary primarily by experimental setup, but commonly use 20–100 μg per dose, administered every other day or as a single dose, in models expressing rat CD28.