Antiviral T cells and CAR T cells in a patient with disseminated Adv infection. (A) Time course of peripheral blood AdV hexon protein–specific CD4⁺ (blue) and CD8⁺ (green) T cells. T cells specific for the AdV hexon protein were identified ex vivo after short-term stimulation of total PBMC using libraries of overlapping peptides covering the complete sequence of the protein (Miltenyi Biotec, catalog no. 130-093-496). Intracellular staining of cytokines followed by flow cytometry served as a readout assay. AdV-specific T cells were defined as interferon gamma–positive CD3⁺ T cells. Black arrows indicate time points when the patient received lymphodepleting (LD) chemotherapy or CAR T-cell therapy. The black dotted line indicates the time point at which the diagnosis of Adv infection was made. (B) CAR T-cell numbers and proportions of CD4⁺ and CD8⁺ CAR-Ts after LD chemotherapy and CAR T-cell infusion. The CAR T cells were identified by staining the expression of the CAR on the cell surface using BCMA CAR detection reagent (Miltenyi Biotec, catalog no. 130-126-090) and costaining with anti-CD3 and other T-cell markers. Samples were acquired using a Miltenyi MACSQuant Analyzer 10 Flow Cytometer. Analysis of Flow cytometry data was performed using FlowJo software (BD Biosciences, San Jose, CA). Black arrows indicate the time points at which the patient received LD chemotherapy or CAR T-cell therapy. The black dotted line indicates the time point at which the diagnosis of Adv infection was made. (C) Course of white blood cell, neutrophil, and lymphocyte counts over time. (D) Dot plots showing peripheral blood AdV hexon protein–specific CD4⁺ (lower row) and CD8⁺ (upper row) T cells at different time points after CAR T-cell infusion. The black dotted line indicates the time point at which the diagnosis of Adv infection was made. (E) Dot plots showing peripheral blood influenza NP protein–specific CD4⁺ (lower row) and CD8⁺ (upper row) T cells at different time points after CAR T-cell infusion. T cells specific for the influenza A (H1N1) NP protein were identified ex vivo after short-term stimulation of total PBMC using libraries of overlapping peptides covering the complete sequence of the protein (Miltenyi Biotec, catalog no. 130-097-278). ALC, absolute lymphocyte count; ANC, absolute neutrophil count; Dx, diagnosis; WBC, white blood cell.