Splenic T-cell commitment. Splenocytes were stimulated with PMA and ionomycin in the presence of Brefeldin A for 4 hours. Cells were first stained for CD3 and CD8. After fixation and permeabilization, intracellular staining for IFN-γ, IL-4, and IL-17 was performed. (A) For FCM analyses, lymphocytes were first gated on their forward (FSC) and side scatter (SSC), then on their expression for CD3 and CD8. Cells were defined as Tc1: CD3⁺CD8⁺IFN-γ⁺, Tc2: CD3⁺CD8⁺IL-4⁺, Tc17: CD3⁺CD8⁺IL-17⁺, Th1: CD3⁺CD8⁻IFN-γ⁺, Th2: CD3⁺CD8⁻IL-4⁺, and Th17: CD3⁺CD8⁻IL-17⁺. The results of a representative RTX-nonresponder patient are depicted. (B) Data are summarized in dot plots representing the expression of each cytokine among T cells, in 9 controls (black circles), 10 RTX-untreated patients (black squares), and 9 RTX nonresponders (black triangles). The horizontal bar represents the median with the interquartile range. P values were derived by Mann-Whitney U test. *P < .05; **P < .01; ***P < .001. NS, nonsignificant. (C) IFN-γ–producing cells were located within periarteriolar lymphoid sheath (PALS) and in the red pulp and consist of CD8⁺ and CD4⁺ T cells (CD8, CD4, and IFN-γ staining, DAB, magnification ×400). Representative immunohistochemistry of 1 RTX-untreated ITP patient.