LFA 3 Human

What is LFA-3 and what is its basic structure?

LFA-3 (CD58) is a 210-222 amino acid protein belonging to the CD2 family of the immunoglobulin superfamily. The mature protein contains an extracellular domain with six N-linked glycosylation sites, followed by a hydrophobic transmembrane region and a short cytoplasmic domain. The mature glycoprotein is heavily glycosylated, with carbohydrate content estimated to comprise 44-68% of the molecule. The protein is encoded by a single gene in humans, producing a 1.3 kb mRNA transcript that is widely distributed across human tissues and cell lines . Unlike many other immunological markers, no mouse or rat homolog of CD58 has been identified, which presents unique challenges for translational research models .

What forms of LFA-3 exist and how are they anchored to the cell membrane?

Human cells can express two distinct forms of LFA-3:

• Transmembrane form: Contains a conventional transmembrane domain and cytoplasmic tail.

• Phosphatidylinositol (PI)-linked form: Attached to the outer leaflet of the plasma membrane via a glycosylphosphatidylinositol anchor.

Both forms are functionally active, but they may have distinct roles. Research with mutant cells deficient in phosphatidylinositol-anchored proteins has demonstrated that these cells express only the transmembrane form of LFA-3. Notably, these studies confirmed that [³H]ethanolamine is not incorporated into LFA-3 of mutant cells, indicating the complete absence of the PI anchor moiety . When the normal biosynthesis of the PI-anchored form is blocked, two intermediate forms accumulate - one with an intact polypeptide chain and another with a truncated chain. The truncated form, lacking membrane attachment, is secreted into culture supernatants .

How does LFA-3 contribute to T cell activation?

LFA-3 provides critical co-stimulatory signals for optimal T cell expansion and activation. T cells require two distinct signals for full activation: (1) engagement of the T cell receptor and (2) co-stimulatory signals through distinct surface molecules. The LFA-3/CD2 interaction represents one of these essential co-stimulatory pathways .

Studies have demonstrated that LFA-3-expressing cells (like LFA-3+ L cells), when combined with anti-CD3 monoclonal antibodies or suboptimal doses of phytohemagglutinin (PHA), effectively stimulate the proliferation of human peripheral blood T cells. This proliferation can be specifically inhibited by monoclonal antibodies directed against either CD2 or LFA-3, confirming the pathway's specificity . Additionally, LFA-3 has been shown to increase expression of activation markers including IL-2 receptor, 4F2, transferrin receptor, and HLA-DR on stimulated thymocytes .

What methodologies can determine whether LFA-3 functions are adhesion-dependent or signaling-dependent?

Distinguishing between LFA-3's adhesion and signaling functions requires specialized experimental approaches:

• Mutant cell studies: Using cells expressing only specific forms of LFA-3 (transmembrane vs. PI-linked) to determine which functions remain intact.

• Antibody blocking experiments: Selectively blocking CD2/LFA-3 interactions while preserving other cellular functions.

• Reconstitution assays: Adding purified forms of LFA-3 (like T11TS, the sheep form of LFA-3) to systems where human LFA-3 has been blocked.

Research employing these approaches has demonstrated that both transmembrane and PI-linked LFA-3 forms can mediate adhesion functions. For example, mutant JY cells expressing only the transmembrane form formed conjugates with cytotoxic T lymphocytes (CTL) and were lysed similarly to wild-type cells expressing both forms. Antibody blocking experiments confirmed the predominant role of the CD2/LFA-3 pathway in these interactions .

What purification methods are most effective for isolating functional LFA-3?

The most effective purification protocol for obtaining functional LFA-3 involves:

• Source material preparation: Typically using Triton X-100 lysates of human erythrocytes, which express high levels of the PI-linked form.

• Immunoaffinity chromatography: Using anti-LFA-3 antibodies (such as TS2/9) coupled to Sepharose CL-4B.

• Elution conditions: Eluting bound LFA-3 at pH 3 in the presence of 1% octylglucoside detergent to maintain protein solubility.

• Aggregation treatment: For some functional studies, aggregated LFA-3 can be prepared through multiple freeze-thaw cycles .

This method yields purified LFA-3 suitable for functional studies, including T cell activation assays and binding studies.

How can researchers effectively block and reconstitute CD2/LFA-3 interactions in experimental systems?

Researchers can manipulate CD2/LFA-3 interactions using the following approaches:

• Selective blocking: Using monoclonal antibodies like G26 that specifically target human LFA-3 and block its interaction with CD2.

• Xenogeneic reconstitution: Adding purified T11TS (sheep LFA-3), which binds human CD2 but is not recognized by antibodies like G26, allowing selective reconstitution of certain pathways.

This strategy has revealed pathway-specific requirements. For example, when the response of peripheral blood mononuclear cells (PBMC) to various T cell mitogens was blocked using the G26 antibody, addition of purified T11TS restored the T cell response to PHA-P but not to ConA, surface aldehydes, anti-CD3 mAb, or allogeneic cells. This indicates that different T cell activation pathways have distinct requirements for LFA-3 co-stimulation .

How do the distinct forms of LFA-3 differ in their functional capabilities?

The two forms of LFA-3 (transmembrane and PI-linked) appear to have overlapping but potentially distinct functions:

Studies with mutant cells expressing only the transmembrane form demonstrate that this form alone can support critical immune functions including CTL conjugate formation, target cell lysis, and stimulation of CTL proliferation. This challenges earlier assumptions that the PI-linked form might be exclusively responsible for certain functions .

What is the relationship between LFA-3 expression and pathological conditions?

Recent research suggests that LFA-3 may play important roles in various pathological conditions:

• Viral infections: Cytomegalovirus infection has been shown to affect CD8 T cells via the CD2-LFA-3 pathway, with potential implications for immune responses .

• HIV infection: The CD2-LFA-3 pathway appears to be involved in CD8 T cell responses during HIV infection .

• Atherosclerosis: Emerging evidence suggests a role for the CD2-LFA-3 pathway in atherosclerosis development and progression .

These findings suggest that targeting the LFA-3/CD2 pathway may have therapeutic potential in various disease contexts.

What are the optimal conditions for studying LFA-3-mediated T cell activation in vitro?

When designing experiments to study LFA-3-mediated T cell activation, researchers should consider:

• Cell source optimization:

  • For primary cells: Freshly isolated peripheral blood T cells or thymocytes

  • For established lines: JY cells (B lymphoblastoid) express both forms of LFA-3 and serve as good model systems

• Activation protocols:

  • LFA-3 at 250 ng/ml final concentration in human A serum

  • Combination with suboptimal doses of mitogens (particularly PHA)

  • Addition of IL-2 (1 U/ml) for some protocols

• Readout systems:

  • Proliferation: [³H]thymidine incorporation after 3-4 days

  • Activation markers: Flow cytometric analysis of IL-2R, 4F2, transferrin receptor, and HLA-DR

  • Functional assays: Cytokine production, cytotoxicity

These conditions have been shown to provide reproducible results in multiple experimental systems .

How should researchers interpret contradictory data regarding LFA-3 function in different experimental systems?

When facing contradictory results regarding LFA-3 function, researchers should consider:

• Form-specific effects: Whether the experimental system predominantly involves the transmembrane or PI-linked form of LFA-3

• Context-dependent function: The LFA-3/CD2 pathway functions differently depending on the activation stimulus (e.g., PHA vs. ConA)

• Cell type differences: Responses may differ between peripheral T cells, thymocytes, and cell lines

• Species differences: The lack of a murine LFA-3 homolog means that mouse models may not accurately reflect human LFA-3 biology

For example, purified T11TS (sheep LFA-3) restores T cell responses to PHA-P but not to ConA, surface aldehydes, or anti-CD3 mAb in systems where human LFA-3 is blocked. This suggests that the co-stimulatory effect of LFA-3 is mechanistically different in PHA-P stimulation compared to other polyclonal T cell activators .

What emerging technologies might advance our understanding of LFA-3 biology?

Several cutting-edge approaches hold promise for deeper insights into LFA-3 function:

• Cryo-electron microscopy: For detailed structural analysis of LFA-3/CD2 interactions at molecular resolution

• CRISPR-Cas9 gene editing: For generating precise mutations in LFA-3 to study structure-function relationships

• Single-cell analysis: To understand heterogeneity in LFA-3 expression and function across different cell populations

• Advanced imaging techniques: Such as super-resolution microscopy to study the spatial organization of LFA-3 in the membrane

These approaches could help resolve long-standing questions about the specific roles of different LFA-3 forms and their mechanisms of action.

What therapeutic applications might target the LFA-3/CD2 pathway?

The fundamental role of LFA-3 in T cell biology suggests several potential therapeutic applications:

• Immunomodulation: Using antibodies or small molecules to block or enhance CD2/LFA-3 interactions

• Cancer immunotherapy: Potentially enhancing T cell responses against tumors by manipulating this pathway

• Autoimmune disease treatment: Blocking pathological T cell activation in conditions like rheumatoid arthritis

• Infection control: Particularly in viral infections where CD2/LFA-3 interactions have been implicated, such as HIV and cytomegalovirus

Development of these applications requires deeper understanding of the pathway's context-specific functions and careful consideration of potential off-target effects.