Cytokine production in PBMCs from XLA patients after TREM-1 triggering. (A) Expression of TREM-1 on PBMCs of healthy controls and of XLA patients was analyzed by flow cytometry. Representative histograms show overlays of staining with isotype-matched (dashed line) or anti–TREM-1 (full line) mAbs gated on CD14⁺ cells. (B) PBMCs were prepared from peripheral blood of XLA patients and healthy individuals by Ficoll-Paque gradient centrifugation. Cells were incubated with plate-bound isotype control or anti–TREM-1 mAbs for 24 hours and TNF-α production was analyzed by ELISA. Because of the lack of B cells in XLA patients, slightly increased frequency of TREM-1⁺ cells was observed (healthy controls: 21.4 ± 5.4%; XLA: 30.3 ± 8.0%). Thus, levels of TNF-α production were normalized to 5 × 10⁴ of CD14⁺ cells. Median value of group is depicted in the graph as a line.