CTLA4 blocking antibodies have shown prominent therapeutic efficacy in melanoma by enhancing antitumor immunity. However, this strategy is accompanied by systemic autoimmune side effects. Previously, we reported the CTLA4-CD28 chimera mutant (CTC28), which consists of the extracellular and transmembrane domains of CTLA4 linked to the cytoplasmic domain of CD28. CTC28-overexpressing T cells convert the inhibitory signal of CTLA4 into a stimulatory signal of CD28 and significantly enhance T cell activity, which leads to markedly improved antitumor effect of T cell therapy without accompanying systemic autoimmunity. However, the expansion of large numbers of tumor antigen-specific T cells for CTC28-modification is technically challenging. Meanwhile, in CAR-T cell therapy, the rapid generation of sufficient number of tumor-specific T cells could be achieved by introducing tumor antigen-specific CAR genes. Therefore, dual modification of T cells with CAR and CTC28 genes may secure a large number of functionally enhanced tumor-specific T cells. In this study, we generated murine CD19-targeting CAR-T cells co-modified with CTC28 (CD19-CTC28 CAR-T) and tested their efficacy in murine B-cell lymphoma models. CD19-CTC28 CAR-T cells showed superior anti-tumor efficacy over CD19 CAR-T cells in vivo, dramatically expanded in peripheral blood, and showed more effector-like phenotype in spleen and tumor-infiltrating CAR-T cells. To further elucidate which cell type provides CD80/86, the ligands for CTC28, in CD19-CTC28 CAR-T cells, we removed CD80/86 on tumor cells which were the most likely cell type. Surprisingly, CD19-CTC28 CAR-T cells still showed significant anti-tumor efficacy against CD80/68-deficient tumors. In the CD80/86 knockout host, this anti-tumor efficacy was partially abrogated and the enhancement of CAR-T cell expansion was completely abolished, suggesting that host antigen-presenting cells provide CD80/86 to the CAR-T cells. Particularly, B cell-expressed CD80/86 was most important for CAR-T cell expansion as demonstrated in B cell-specific CD80/86 KO hosts. Nevertheless, CD19-CTC28 CAR-T cells in CD80/86 knockout hosts still showed partial efficacy, which completely disappeared when CD80/86 knockout CAR-T cells were used, implying that unexpected expression of CD80/86 on CAR-T cells mediated this efficacy. Interestingly, this efficacy was mediated by both trans-endocytosis of CD80/86 by CTC28 and CTC28 signaling in the CAR-T cells. Finally, the enhanced effect of CD19-CTC28 CAR-T was recapitulated in a human B-cell lymphoma model performed in NSG immunodeficient mice. Thus, CAR and CTC28 dual-modified T cell therapy could be a promising strategy for reinforced anti-tumor T cell therapy using unexpected mechanisms.