Altered CXCR4 internalization in WHIM¹⁰¹³ lymphocytes. (A) Cell surface expression levels of CXCR4 in CD4⁺-gated T cells from PBMCs of a healthy subject (control, left panel) or WHIM¹⁰¹³ patient P1 (right panel). CXCR4 levels were assessed using the 12G5 (empty histograms) or isotype control (CTRL, gray histograms) mAb. (B) CXCR4 cell surface expression in CD4⁺-gated T cells from PBMCs of WHIM¹⁰¹³ patients P1 (▦) and P2 (▪) or healthy subjects (□). ^*P < .05 and ^**P < .005 compared with healthy subjects. (C) Time course of CXCL12-promoted CXCR4 endocytosis in CD4⁺-gated T cells from patients P1 (▦) and P2 (▪) versus healthy subject (□). (D) CXCR4 cell surface expression in A0.01 T cells (left panel) or PBMCs from healthy individuals (right panel) nontransduced (NT) or transduced with the indicated CXCR4 variant receptors. □ indicates +CXCL12; ▪, +PMA. In untreated A0.01 T cells, the geometric MFI of CXCR4^wt, CXCR4¹⁰⁰⁰, and CXCR4¹⁰¹³ receptors were 30, 35, and 28, respectively. Analysis in PBMCs was assessed in CD4⁺-gated T cells. ^*P < .05 and ^**P < .005 compared with CXCR4^wt-expressing A0.01 T cells or with NT T lymphocytes. Results, expressed as percentage of untreated cells, are from 3 independent experiments (mean ± SEM) (B,D) or from 1 representative experiment of 2 (C). (E) Cell surface expression of T7-GFP-CXCR4^wt in CXCR4^wt or CXCR4¹⁰¹³ CHO cells either untreated (dot plot, top) or treated with CXCL12 (bottom). □ indicates CXCR4^wt; ▪, CXCR4¹⁰¹³. In untreated CHO cells, the geometric MFI of CXCR4^wt GFP^-/gate A), CXCR4¹⁰¹³ GFP^- (gate B), CXCR4^wt GFP⁺ (gate C), and CXCR4¹⁰¹³ GFP⁺ (gate D) were 47, 57, 63, and 61, respectively. Expression of T7-GFP-CXCR4^wt is roughly comparable when coexpressed with CXCR4^wt (geometric MFI = 150, gate E) or CXCR4¹⁰¹³ (geometric MFI = 130, gate F). Analysis of CXCL12-promoted receptor endocytosis was performed in the cell gates defined earlier in the legend. Results (mean ± SEM) are representative of 2 determinations and are expressed as percentage of untreated cells. (F) Cell surface expression of T7-GFP-CXCR4^wt in CD4⁺-gated T cells from PBMCs of a healthy individual transfected with CXCR4^wt or CXCR4¹⁰¹³ variant either untreated (dot plot, upper panel) or treated with CXCL12 or PMA (lower panel). □ indicates CXCR4^wt; ▪, CXCR4¹⁰¹³. In untreated CD4⁺-gated T cells, the geometric MFI of CXCR4 in gates A, B, C, and D were 195, 210, 620, and 580, respectively. Expression of T7-GFP-CXCR4^wt is roughly comparable when coexpressed with CXCR4^wt (geometric MFI = 260, gate E) or CXCR4¹⁰¹³ (geometric MFI = 220, gate F). Analysis of CXCL12- or PMA-promoted receptor endocytosis was performed in cell gates defined above. Results are from 1 representative experiment of 2 and are expressed as percentage of untreated cells.