Characterization of CD18 expression, ITGB2 gene mutations, T-cell receptor repertoire, and T-cell functions. (A) Analysis of CD18 expression. Shown are the results of CD18 expression on granulocytes, monocytes, and lymphocytes. Open peaks indicate control Ab; solid peaks represent anti-CD18 mAb. (B) CD18 expression on the patient's lymphocyte subsets. The percentage of cells gated in each quadrant is shown. (C) Mutation analysis. The ITGB2 gene was amplified from DNA extracted from normal PBMCs, PBMCs from the parents, and the patient's granulocytes and CD18⁺ T cells. Direct sequencing was performed using an automated sequencer. Bars show the locations of the mutations. (D) Microsatellite analysis. Three different markers were amplified with FAM-labeled specific primers and subjected to GeneScan analysis. HUMARA indicates human androgen receptor gene; HO-1, heme oxygenase-1. (E) Expression profiles of TCR VB subfamilies. Peripheral blood samples were stained with mAbs for individual TCR VB subfamilies together with anti-CD8 and anti-CD18 mAbs. The expression of each TCR VB by CD8⁺ or CD18⁺CD8⁺ T cells was analyzed by FACS. (F) CDR3 spectratyping. A TCR VB22 fragment was amplified from cDNA with specific primers. The size distribution of polymerase chain reaction products was determined by GeneScan analysis. (G) Analysis of CD2-mediated T-cell activation. PBMCs from healthy control subjects and the patient were cultured for 16 hours with a combination of anti-CD2 mAbs alone or together with interleukin-2 or anti-CD28 mAb. CD69 and CD25 expression was evaluated in the patient's CD18⁻CD8⁺ T (□) and CD18⁺CD8⁺ T (⊡) cells. ▪ indicate control CD8⁺ T cells from healthy adults. Error bars represent SD.