CD28 costimulation enhances the expansion and persistence of chimeric antigen receptor (CAR)-modified T cells in lymphoma patients. This study compared two CAR constructs in patients with B-cell lymphomas: one with CD28 and ζ endodomains, and another with only the ζ endodomain. CAR+ T cells with the CD28 endodomain showed significantly greater expansion and persistence compared to those without. These results demonstrate the advantage of CARs with dual signal domains and confirm a method for comparing CAR-modified T cells within individual patients, reducing variability between patients and accelerating the development of optimal T cell immunotherapies. The study involved six patients with relapsed or refractory non-Hodgkin lymphoma (NHL). Each patient received two autologous T cell products expressing CARs specific for CD19. One product included the CD28 and ζ endodomains, while the other included only the ζ endodomain. CAR+ T cells with the CD28 endodomain showed enhanced expansion and persistence in peripheral blood, as measured by quantitative PCR (Q-PCR) assays. These cells also persisted longer and retained the ability to expand upon re-stimulation. In contrast, CAR+ T cells with only the ζ endodomain showed reduced expansion and became undetectable after six weeks. Tissue analysis of a skin lesion from one patient revealed that CAR.CD19-28ζ+ T cells were present in the tumor, suggesting that the enhanced expansion and persistence in peripheral blood may extend to tumor sites. However, despite the improved T cell responses, the clinical benefits were not sustained in all patients. The study highlights the importance of CD28 costimulation in enhancing T cell function and persistence, but further strategies may be needed to achieve long-term clinical benefits. The method used in this study allows for direct comparison of CAR-modified T cells within individual patients, reducing variability and accelerating the development of optimal T cell immunotherapies.CD28 costimulation enhances the expansion and persistence of chimeric antigen receptor (CAR)-modified T cells in lymphoma patients. This study compared two CAR constructs in patients with B-cell lymphomas: one with CD28 and ζ endodomains, and another with only the ζ endodomain. CAR+ T cells with the CD28 endodomain showed significantly greater expansion and persistence compared to those without. These results demonstrate the advantage of CARs with dual signal domains and confirm a method for comparing CAR-modified T cells within individual patients, reducing variability between patients and accelerating the development of optimal T cell immunotherapies. The study involved six patients with relapsed or refractory non-Hodgkin lymphoma (NHL). Each patient received two autologous T cell products expressing CARs specific for CD19. One product included the CD28 and ζ endodomains, while the other included only the ζ endodomain. CAR+ T cells with the CD28 endodomain showed enhanced expansion and persistence in peripheral blood, as measured by quantitative PCR (Q-PCR) assays. These cells also persisted longer and retained the ability to expand upon re-stimulation. In contrast, CAR+ T cells with only the ζ endodomain showed reduced expansion and became undetectable after six weeks. Tissue analysis of a skin lesion from one patient revealed that CAR.CD19-28ζ+ T cells were present in the tumor, suggesting that the enhanced expansion and persistence in peripheral blood may extend to tumor sites. However, despite the improved T cell responses, the clinical benefits were not sustained in all patients. The study highlights the importance of CD28 costimulation in enhancing T cell function and persistence, but further strategies may be needed to achieve long-term clinical benefits. The method used in this study allows for direct comparison of CAR-modified T cells within individual patients, reducing variability and accelerating the development of optimal T cell immunotherapies.