Homotypic cell aggregation via conformational change of CD44 molecule induced by anti-CD44 monoclonal antibodies

Area of Science:

• Immunology
• Cell Biology
• Hematology

Background:

• Leukocyte homotypic cell aggregation is a key adhesive event triggered by cellular activation.
• The precise mechanisms underlying CD44 monoclonal antibody (mAb)-induced homotypic cell aggregation remain incompletely understood.
• CD44 is implicated in various cellular processes, including cell adhesion and migration.

Purpose of the Study:

• To investigate the mechanism of homotypic cell aggregation induced by different anti-CD44 monoclonal antibodies (mAbs).
• To characterize the binding sites and functional effects of novel anti-CD44 mAbs.
• To elucidate the cellular requirements for CD44-mediated leukocyte aggregation.

Main Methods:

• Utilized four distinct anti-CD44 mAbs (TL-1, OS/37, BU52, Hermes-3) targeting different epitopes.
• Assessed homotypic cell aggregation in various hematopoietic cell lines and normal peripheral blood lymphocytes (PBL).
• Employed blocking assays with antibodies against adhesion molecules (LFA-1, ICAM-1, VLA-4, L-selectin) and anti-metabolic agents.
• Investigated energy dependence and protein synthesis requirements using cycloheximide and actinomycin D.
• Analyzed CD44 expression levels via Fluorescence-Activated Cell Sorting (FACS).

Main Results:

• The novel anti-CD44 mAb TL-1, binding a non-hyaluronate site, induced strong homotypic cell aggregation in multiple cell types, including a B cell line from leukocyte adhesion deficiency syndromes (LAD) patients and PBL.
• Other mAbs (OS/37, BU52) targeting hyaluronate binding sites induced weaker aggregation.
• TL-1-induced aggregation was blocked by mAbs recognizing non-hyaluronate (Hermes-3) or hyaluronate (OS/37) binding sites, but not BU52.
• Aggregation was energy-dependent, associated with actin filaments, and did not require de novo protein synthesis.
• TL-1 binding did not alter CD44 surface expression intensity.

Conclusions:

• CD44-mediated homotypic cell aggregation can be induced by mAbs targeting distinct epitopes, including non-hyaluronate binding sites.
• The aggregation process is an active, energy-dependent cellular event involving cytoskeletal rearrangements.
• These findings provide novel insights into the complex mechanisms of CD44-mediated leukocyte adhesion and aggregation.