[Phenotypic and functional changes in endogenous T cells during the bystander effect in different disease states of lymphomas treated with CD19-targeted chimeric antigen receptor T cells]

Objective: To investigate the bystander effect of endogenous T cells within the tumor microenvironment, as well as their roles and phenotypic changes during remission and relapse in mice with B-cell lymphoma receiving CD19-targeted chimeric antigen receptor T (CAR-T) cell therapy. Methods: At the Institute of Hematology, Xuzhou Medical University, in vitro experiments and mouse models were established using BALB/c mice to evaluate the efficacy of CD19 CAR-T cells in treating CD19(-) and CD19(+)A20 lymphomas. In the in vitro experiments, the mice were categorized into the following groups based on the type of co-cultured cells: activated T cell (n=3) and CD19-targeted CAR-T cell (n=3) groups. In the CD19(-) A20 lymphoma mouse model, the mice were assigned to the activated T-cell (n=5) and CD19 CAR-T cell treatment (n=5) groups based on the type of cells used for treatment. For the CD19(+) A20 lymphoma mouse model, the mice were divided into the activated T cell treatment (n=3-8), CD19 CAR-T cell treatment (n=14), remission (clinical score ≤ 1), and relapse (clinical score >1) groups. Therapeutic efficacy was evaluated, and flow cytometry was used to analyze the subsets, activation status, and cytokine secretion profiles of endogenous T cells at different disease states. Results: In vitro experiments demonstrated that, compared with the activated T-cell group, CD19 CAR-T cells exerted no specific cytotoxic effect on CD19(-) A20 cells [survival rates: (40.83±1.70) %vs (39.07±1.40) %, P=0.238]. However, in immunocompetent mice, compared with the activated T-cell treatment, CD19-targeted CAR-T cell therapy resulted in reduced weight [ (0.14±0.10) vs (0.81±0.24) g, P=0.002] and significantly diminished subcutaneous CD19(-) A20 tumor volume [ (335.40±130.20) vs (542.00±302.40) mm(3), P=0.004], accompanied by increased infiltration of endogenous CD3(+) T cells [ (36.34±6.53) % vs (9.49±9.42) %, P=0.001]. CD8(+) T cells exhibited increased CD69 expression [ (21.38±4.85) % vs (3.15±1.16) %, P=0.001] and a trend toward decreased expression of exhaustion-associated molecules PD-1 and TIGIT (all P value >0.05). In the mouse model of CD19(+) A20 lymphoma remission and relapse, upon disease relapse, the proportion of endogenous CD8(+) T cells in the spleen (SP) and bone marrow (BM) showed a downward trend during disease relapse compared with that during the remission period, the CD69 expression intensity decreased (CD4(+) T cell: BM: 733.20±274.40 vs 1 093.00±131.20, P=0.013; SP: 290.50±142.40 vs 478.70±63.94, P=0.018; CD8(+) T cell: BM: 1 223.00±391.50 vs 1 892.00±540.40, P=0.041; SP: 342.30±258.10 vs 500.00±113.90, P=0.325) and the secretion levels of cytokines IFN-γ, TNF-α, GM-CSF, and granzyme B were all diminished (all P value <0.05). During relapse, the proportion of effector T cell (Teff) cells decreased significantly, whereas the proportions of naive T cell (Tn) cells and memory T cell (Tm) cells increased significantly (all P value <0.05) . Conclusion: The specific cytotoxic activity of CD19-targeted CAR-T cells in vivo can activate endogenous T cells and induce a bystander effect, which non-specifically inhibits lymphoma growth. However, following disease relapse, the cytotoxic function of endogenous T cells gradually diminishes and shifts toward resting and memory phenotypes.