Abnormal CXCR4 expression and intracellular detection of CXCL12 in ICL CD4⁺ T lymphocytes. (A) Proportion of CD3⁺ CD4⁺ T cells in whole blood sample from a representative ICL patient (P1) as determined by flow cytometry. (B) Membrane expression levels of endogenous CCR5 and CXCR4 in CD4⁺ T cells from P1 (expressed as %). Whole blood recovered before (untreated) or after (in vivo) 5 days of therapeutic administration of IL-2 (4.5 million units twice a day subcutaneously) or PBMCs incubated overnight with 10 UI/mL IL-2 (in vitro) were labeled using FITC-conjugated CCR5 and PE-conjugated CXCR4 mAbs. Background fluorescence was measured using the corresponding isotype control Ab. The inset summarizes the proportion of CXCR4⁺ CD4⁺ T cells in the 3 patients (P1, P2, and P6) before and after IL-2 therapy. Mean CXCR4 expression is reported before and after the first 5-day cycle of IL-2 (P < .001). (C) Intracellular staining of CXCL12 in CD4⁺-gated T cells from PBMCs of a healthy (H1) or ICL (P1) subject obtained before (untreated) or after (in vivo) 5 days of IL-2 administration. Staining was done using an anti–human CXCL12 mAb followed by a FITC-conjugated anti–mouse Ab. The inset summarizes the proportion of CXCL12-containing CD4⁺ T cells in the 3 patients before and after administration of the first cycle of IL-2.