Analyses for CCR7/EBI1 expression.

(A) Reverse transcriptase-polymerase chain reaction (RT-PCR) analysis for CCR7/EBI1 expression. PCR amplification of the CCR7/EBI1 and β-actin–specific products in control CD4⁺CD45RO⁺ T cells (lane 1), freshly isolated adult T-cell leukemia (ATL) cells without (lanes 2, 3, and 4; patients 7, 10, and 6, respectively) and with lymphoid organ involvement (lanes 5, 6, 7, and 8; patients 27, 26, 24, and 23, respectively) was performed for 28 PCR cycles. (B) RT-PCR analysis for CCR7/EBI1 expression in freshly isolated ATL cells from patients with and without lymphoid organ involvement. CCR7/EBI1 expression was evaluated with the ratio of CCR7/β-actin–specific product for control CD4⁺CD45RO⁺ T cells obtained from the peripheral blood of 8 healthy volunteers (A, closed squares), ATL cells from 12 patients without lymphoid organ involvement (B, open circles; patients 1 through 12), and ATL cells from 16 patients with lymphoid organ involvement (C, open circles; patients 13 through 28). RT-PCR analyses were performed independently in triplicate. (C) Flow cytometric analysis for CCR7/EBI1 expression on freshly isolated ATL cells from patients with and without lymphoid organ involvement. Flow cytometric analysis of control CD4⁺CD45RO⁺ T cells and freshly isolated ATL cells was performed with anti-CCR7 mAb, CCR7.6B3. Each point shows the mean fluorescence intensity (MFI) for CCR7/EBI1 staining, which was corrected by substrating MFI obtained with the nonbinding control IgG₁ mouse mAb. Each point of group A indicates the MFI for CCR7/EBI1 staining of CCR7/EBI1-positive CD4⁺CD45RO⁺ T cells because CCR7/EBI1-positive cells were 55% ± 14% of control CD4⁺CD45RO⁺ T cells isolated from 8 healthy volunteers. The bars indicate the mean value of each group; the statistical analysis was performed using Student t test. (D) Flow cytometric analysis for CXCR4 expression on freshly isolated ATL cells from patients with and without lymphoid organ involvement. Flow cytometric analysis of control CD4⁺CD45RO⁺ T cells and freshly isolated ATL cells was performed with anti-CXCR4 mAb. Each point shows the MFI for CXCR4 staining, which was corrected by substrating MFI obtained with the nonbinding control IgG_2amouse mAb. Each point of group A indicates the MFI for CXCR4 staining of CXCR4-positive CD4⁺CD45RO⁺ T cells because CXCR4-positive cells were 76% ± 18% of control CD4⁺CD45RO⁺ T cells isolated from 8 healthy volunteers. The bars indicate the mean value of each group; the statistical analysis was performed using Student t test.