Changes in T-cell immunity. (A) Changes in cytokine profile of T cells. PBMCs from pre- and posttherapy time points were thawed and stimulated with PMA and ionomycin. The percentage of CD3⁺ T cells expressing individual cytokines (IFNγ, IL2, IL17, and IL4) was evaluated using intracellular cytokine staining and flow cytometry. Shown is the fold change in cytokine production on days 7 and 14 of cycle one as well as before starting cycle 2 (PreC2) compared with cytokine production by T cells before starting therapy (preC1). (B-C) PBMCs from baseline (pretreatment) were stimulated overnight with an overlapping peptide library derived from SOX2 (B) or a pool of peptides from viral antigens (CEF) as a control (C). Reactivity to the peptide pools was assayed with the detection of IP10 in the supernatant. Stimulation index refers to fold increase in IP10 over control wells. (D) Changes in SOX2 reactivity during therapy in a patient P2 with clinical response to therapy. PBMCs were stimulated overnight with peptide pools derived from SOX2 as in panel B. After overnight culture, the presence of IP10 in supernatant was assayed using Luminex.