Flow cytometry–based immunologic profiling: representative flow plots and gating strategy. (A) Gating strategy by flow cytometry for the detection of CD4⁺ and CD8⁺ T cells (singlet [forward scatter A{FSC-A} × FSC-H diagonal] live [Aqua] CD3⁺ lymphocytes) extracted from rectosigmoid biopsy tissue (RB, top panels) and from peripheral blood (PB, bottom panels). (B) Detection of CD38 and Ki67 expression in CD8⁺ T cells (left) as well as FoxP3 and Ki67 in CD8⁺ and CD4⁺ T cells from rectosigmoid biopsy tissue (top) and from peripheral blood (bottom). (C-D) Antigen-specific CD8⁺ (top) CD4⁺ (bottom) T-cell responses in peripheral blood after stimulation with PBS (mock), CMV, HIV, Candida albicans (C alb), or SEB antigens, as measured by CFSE^low proliferative responses (C) or IFNγ intracellular cytokine detection (D). (E) Detection of intracellular cytokines (IFNγ, IL-2, IL-17, or TNFα) in CD4⁺ T cells from rectosigmoid biopsy tissue (top) and from peripheral blood (bottom), after PMA/ionomycin stimulation in vitro. All of these analyses were carried out using cells from a representative noncontroller (1256).