Project description:To gain further insight into how CCT fusion engineering impacts their function, we characterized CAR-T cells using transcriptome profiling. We performed mRNA-seq from all four groups of CAR-T cells, either without stimulation (baseline) or with 2 rounds of NALM6GL stimulation. For CAR-T cells that were cocultured with 2 rounds of NALM6GL cells, we sorted the pure CAR-T populations (CAR+; GFP-) before subjecting them to RNA sequencing
Project description:To systematically investigate the effect of CCT engineering on CAR-T cell phenotypes in vivo, we used single-cell RNA sequencing (scRNA-seq) to characterize the full spectrum of engineered CAR-T cells and their transcriptomic profiles
Project description:In our previous studies, using a B cell vaccine (scFv-Her2), the targeting of tumor-associated antigen Her2 (human epidermal growth factor receptor-2) to B cells via the anti-CD19 single chain variable fragment (scFv) was shown to augment tumor-specific immunity, which enhanced tumor control in the prophylactic and therapeutic setting. However, the fusion protein displayed limited activity against established tumors, and local relapses often occurred following scFv-Her2 treatment, indicating that scFv-Her2-induced responses are inadequate to maintain anti-tumor immunity. In this study, targeting the IV region (D4) of the extracellular region of Her2 to B cells via CD19 molecules (scFv-Her2D4) was found to enhance IFN-γ-producing-CD8+ T cell infiltration in tumor tissues and reduced the number of tumor-infiltrating myeloid-derived suppressor cells (MDSCs). However, negative co-stimulatory molecules such as programmed cell death protein-1 (PD-1), CD160, and LAG-3 on T cells and programmed death protein ligand-1 (PD-L1) on tumor cells were upregulated in the tumor microenvironment after scFv-Her2D4 treatment. Further, anti-PD1 administration enhanced the efficacy of scFv-Her2D4 and anti-tumor immunity, as evidenced by the reversal of tumor-infiltrating CD8+ T cell exhaustion and the reduction of MDSCs and Treg cells, which suppress T cells and alter the tumor immune microenvironment. Moreover, combining this with anti-PD1 antibodies promoted complete tumor rejection. Our data provide evidence of a close interaction among tumor vaccines, T cells, and the PD-L1/PD-1 axis and establish a basis for the rational design of combination therapy with immune modulators and tumor vaccine therapy.
Project description:Thermal ablation to destroy tumor tissue may help activate tumor-specific T cells by elevating the presentation of tumor antigens to the immune system. However, the antitumor activity of these T cells may be restrained by their expression of the inhibitory T-cell coreceptor CTLA-4, the target of the recently U.S. Food and Drug Administration-approved antibody drug ipilumimab. By relieving this restraint, CTLA-4-blocking antibodies such as ipilumimab can promote tumor rejection, but the full scope of their most suitable applications has yet to be fully determined. In this study, we offer a preclinical proof-of-concept in the TRAMP C2 mouse model of prostate cancer that CTLA-4 blockade cooperates with cryoablation of a primary tumor to prevent the outgrowth of secondary tumors seeded by challenge at a distant site. Although growth of secondary tumors was unaffected by cryoablation alone, the combination treatment was sufficient to slow growth or trigger rejection. In addition, secondary tumors were highly infiltrated by CD4(+) T cells and CD8(+) T cells, and there was a significant increase in the ratio of intratumoral T effector cells to CD4(+)FoxP3(+) T regulatory cells, compared with monotherapy. These findings documented for the first time an effect of this immunotherapeutic intervention on the intratumoral accumulation and systemic expansion of CD8(+) T cells specific for the TRAMP C2-specific antigen SPAS-1. Although cryoablation is currently used to treat a targeted tumor nodule, our results suggest that combination therapy with CTLA-4 blockade will augment antitumor immunity and rejection of tumor metastases in this setting.
