CXCR4 is required for TCR-initiated production of IL-2, IL-4, and IL-10. (A) Purified, human PBMC T cells were stimulated with 1 μg/mL plate-bound OKT3 and soluble CD28, cultured for 24 hours, and harvested for analysis via qRT-PCR for CXCL12 mRNA transcript levels. A human bone marrow mesenchymal stromal stem cell line (BMSC) was used as a positive control for CXCL12 expression. The results shown are normalized to the reference gene GAPDH, where GAPDH is set to 100. Each point denotes the mean mRNA transcript level ± standard deviation (SD) for 5 independent donors (n = 2 for BMSC samples). (B-E) Human PBMC T cells were purified, transfected with either control siRNA, a pool of CXCR4 siRNAs (CXCR4 siRNA-1) or CXCR4 siRNA-2 (single siRNA), and cultured for 24 hours. (B-C) CXCR4 cell surface levels were assayed via flow cytometry. Mean fluorescent intensities (MFI) are shown for a representative experiment. (C) Summarizes the results with each bar denoting the mean ± standard error of the mean (SEM). *Significantly different from control siRNA-transfected cells (P < .05; n = 7-9). (D-E) Twenty-four hours after transfection, cells were stimulated as in panel A, then cultured for an additional 24 hours prior to harvest of supernatants for cytokine analysis. Results from 7 to 9 donors, with the black line denoting the average of all donors tested ± SEM. *Significant difference compared with control siRNA-transfected cells (P < .05).