CXCR4¹⁰¹³-mediated chemotaxis depends on the SHSK motif of the receptor. (A) A0.01 T cells were transduced to express identical amounts of CXCR4^wt, CXCR4¹⁰¹³, or the receptors lacking the SHSK motif, CXCR4^wt/Δi3, or CXCR4^1013/Δi3. The panel shows typical cell-surface expression levels of CXCR4^wt (solid line) and CXCR4¹⁰¹³ (dotted line), as assessed by flow cytometric analysis using PE-conjugated 12G5, compared with parental cells (filled peak). The inset depicts the expression levels of CXCR4¹⁰¹³ (▩), CXCR4^wt/Δi3 (■), and CXCR4^1013/Δi3 (▧) compared with that of CXCR4^wt arbitrarily set at 100% (□). Data are means plus or minus SEM (n = 3). (B) Transduced cells were subjected to CXCL12-induced chemotaxis as in Figure 1C. The transmigrated cells recovered in the lower chamber were counted by flow cytometry. The data (means ± SEM; n = 3) represent chemotactic indexes that were calculated as follows: (number of cells that migrated toward CXCL12) / (number of cells that migrated spontaneously). Spontaneous migrations were in the same range for all cell populations, reaching 4.4% plus or minus 1.8%, 3.8% plus or minus 2.2%, 4.2% plus or minus 2.2%, and 3.6% plus or minus 0.6% of input CXCR4^wt-, CXCR4¹⁰¹³-, CXCR4^wt/Δi3-, and CXCR4^1013/Δi3-expressing cells, respectively. As also shown in the panel, pretreating CXCR4^wt- or CXCR4¹⁰¹³-expressing cells with Bordetella pertussis toxin (PTX) at 0.5 μM for 90 minutes abrogated CXCL12-dependent migration. *P < .05 compared with cells with the other receptors in unpaired one-tailed Student t test.