WHIM-mutant CXCR4 recruitment to the T-APC synapse is impaired by competing external CXCL12 and restored by AMD3100. (A) Primary CD4⁺ T cells from healthy donor peripheral blood transfected with EGFP-CXCR4 (WT) or EGFP-CXCR4 (WHIM)–formed conjugates for 15′ with unpulsed or superantigen (sAg)-pulsed EBV-B cells, in the presence or absence of 5 nM to 100 nM CXCL12 and 12.6 μM AMD3100. The cells were fixed, imaged in confocal microscopy, and the RRI to the T-APC synapse for EGFP-CXCR4 was calculated. The graph displays the percentage of cells with CXCR4 recruited to the synapse (ie, with RRI above the mean RRI of control WT T cells incubated with unpulsed EBV-B). The RRI data set for all analyzed cells (n ≥40 per condition) is shown in supplemental Figure 3. Statistical analysis was performed on the proportion of cells displaying recruitment versus nonrecruitment of CXCR4 to the synapse. *P < .05; ***P < .001 (Fisher’s exact test). (B) Representative confocal microscopy images showing the recruitment of EGFP-CXCR4 (WT) or EGFP-CXCR4 (WHIM) to the T-APC synapse. n.s., not significant.