CD19-CD28 is not superagonistic and relies on signal 1. (A) Activity of human PBMCs in response to TGN1412 and CD19-CD28. PBMCs isolated from buffy coats of 3 donors were stimulated with TGN1412 or CD19-CD28 (dose titration, 0-500 nM) and cytokine release was analyzed after 48 hours via multiplex analysis. Bars show mean + SEM of technical triplicates from 3 donors. (B) Experimental design of in vivo cytokine release evaluation. Non–tumor bearing humanized NSG mice (3 mice per group) were treated IV with vehicle (histidine buffer), CD19-CD28 (10 or 1 mg/kg), TGN1412 huIgG4 (10 mg/kg), or TGN1412 msIgG1 (10 mg/kg). (C) Multiplex analysis of serum cytokines at indicated time points after treatment. The absolute cytokine values are shown in supplemental Table 1. (D) Human PBMCs from 4 healthy donors were stimulated with glofitamab (0, 1, 10, or 100 pM) and increasing concentrations of CD19-CD28 (0-200 nM). Upper row, the percentage of CD25 expression on CD8⁺ T cells was analyzed by flow cytometry after 72 hours. Bars show mean + SEM of technical triplicates for each donor. Dots show individual values. Lower row, multiplex analysis of IL-6 in culture supernatants after 48 hours. Bars show mean + SEM of technical triplicates. Dots show individual values. The shades of gray correspond to different donors, with each donor consistently represented by the same shade across all treatment conditions.