A CD2-green fluorescence protein-transgenic mouse reveals very late antigen-4-dependent CD8⁺ lymphocyte rolling in inflamed venules

Intravital microscopy allows detailed analysis of leukocyte trafficking in vivo, but fails to identify the nature of leukocytes investigated. Here, we describe the development of a CD2-enhanced green fluorescence protein (EGFP)-transgenic mouse to characterize lymphocyte trafficking during inflammation in vivo. A CD2-EGFP plasmid construct including the CD2 promoter, the EGFP transgene, and the CD2 locus control region was injected into B6CBA/F₁ pronuclei. EGFP⁺ offspring were backcrossed into C57BL/6 mice for six generations. Flow cytometry demonstrated that all peripheral blood EGFP⁺ cells were positive for CD2 and negative for the granulocyte Ag Ly 6-G (GR-1). EGFP^high cells stained positive for CD2, CD3, CD8, TCR β-chain, and NK1.1 but did not express the B cell and monocyte markers CD45RA, CD19, and CD11b. In vitro stimulation assays revealed no difference in lymphocyte proliferation and IL-2 secretion between EGFP⁺ and EGFP^- mice. Intravital microscopy of untreated or TNF-α-treated cremaster muscle venules showed EGFP⁺ cells in vivo, but these cells did not roll or adhere to the vessel wall. In cremaster muscle venules treated with both TNF-α and IFN-γ, EGFP^high cells rolled, adhered, and transmigrated at a rolling velocity slightly higher (11 μm/s) than that of neutrophils (10 μm/s). Blocking α₄ integrin with amAb increased rolling velocity to 24 μm/s. These findings show that CD8⁺ T cells roll in TNF-αIFN-γ-pretreated vessels in vivo via an α₄ integrin-dependent pathway.