Efficient CCR5 down-regulation in human CD4⁺ T lymphocytes in multiple lymphoid organs. (A) The level of CCR5 expression was compared in EGFP⁺ and mCherry⁺ human CD4⁺/CD45⁺ lymphocytes in lymphoid tissues from multiple transplanted mice. We normalized CCR5 expression level using the mean CCR5 expression in mCherry⁺ cells from peripheral blood (PB) as 1. Samples were obtained between 14 and 20 weeks after intravenous CD34⁺ cell injection. Bar represents mean value; n indicates number of samples. Aggregate difference, comparing CCR5 expression in EGFP⁺ versus mCherry⁺ cells in all tissues, was statistically significant by Student t test (P < .001). (B) A representative gating scheme. (i) The human CD4⁺/CD45⁺ population was identified in the gated lymphocyte population in spleen. (ii-iii) EGFP and mCherry expression was identified in the CD4⁺/CD45⁺ gated population. (iv) mCherry⁻ population was further gated to analyze CCR5 expression in EGFP⁺ and EGFP⁻ population. (v) EGFP⁻ population was further gated to analyze CCR5 expression in mCherry⁺ and mCherry⁻ population. (vi) The mean fluorescent intensity of CCR5 expression was compared in EGFP⁺ and mCherry⁺ population. (C-D) Representative data showing CCR5 down-regulation in multiple lymphoid tissues from a mouse with the highest CCR5 basal expression. (E) Additional dataset from mouse reconstituted with different donor. Data were analyzed as shown in panel B.