Protocol schema and assessment of target antigen expression CAR22 therapy. (A) Overview of the trial design, including enrollment, manufacturing of autologous CD22-directed CAR T cells, administration of the therapy, and follow-up monitoring. Patients had safety assessments performed and blood samples drawn at each arrow after infusion, for assessment of correlative biomarkers, and underwent clinical and radiographic response assessment at each blue arrow. The green arrow indicates the time period during which CAR-FACS, RT-PCR, and cytokines were collected at all specified time points. (B) Gating strategy and (C) flow cytometry histograms of CD19 and CD22 expression for each patient before CAR22 therapy. After CAR19 therapy, P1 demonstrated preserved CD19 expression, whereas P2 had heterogeneous and downregulated CD19 expression. In all 3 patients, CD22 expression was preserved at high levels. (D) Serial biopsy specimens of P3 showing CD19 downregulation after CAR19 therapy, then complete loss of CD19 expression after CAR20.19 therapy. Original IHC image magnification ×40. Anti-CD19 antibody clone BT51E (murine monoclonal, Leica #PA0843). Anti-CD22 antibody clone FPC1 (murine monoclonal, Leica #PA0249). CAR19, anti-CD19 CAR T-cell therapy; CAR20.19, anti-CD19, anti-CD20 tandem CAR T-cell therapy; CAR-FACS, high dimensional flow cytometry immunophenotyping of peripheral blood, including identification of CAR⁺ cells; DLT, dose-limiting toxicity; FSC-A, forward scatter area; IHC, immunohistochemistry; MNC, mononuclear cell; PET-CT, composite positron emission tomographic-computed tomographic imaging; qRT-PCR, quantitative reverse transcriptase-polymerase chain reaction for identification of CAR transgene copies in peripheral blood; SAE, severe adverse event; SSC-A, side scatter area.