Effect of CD73 on immune escape via tryptophan metabolic reprogramming in diffuse large B-cell lymphoma.

7068 Background: Immunotherapy is playing an increasingly important role in patients with relapsed or refractory DLBCL. Our previous studies have shown that high CD73 expression in DLBCL tumor cells correlates with poor prognosis and mediates immune escape. However, the underlying mechanism of CD73-mediated immune escape in DLBCL remains unclear. Methods: We analyzed RNA-Seq data from TCGA and de novo DLBCL patients at our center, comparing CD73 expression levels to identify enriched pathways related to tumor immune escape. These findings were validated in an syngeneic mouse model. To explore the mechanism by which CD73 regulates immune escape in DLBCL, we performed RNA sequencing and targeted tryptophan metabolism sequencing in cell lines. We further conducted co-culture, flow cytometry, Co-IP, ChIP-qPCR in vitro, and generated a mouse subcutaneous xenograft model for in vivo validation. Results: The syngeneic mouse model showed that CD73 knockdown significantly reduced tumor size compared to the control group, while increasing the infiltration of CD8 + T cells and effector CD8 + T cells in tumor tissue, and reducing CD8 + T cell depletion. After systemic CD8 + T cell depletion, the anti-tumor effect of CD73 monoclonal antibody was significantly weakened. RNA-seq and targeted tryptophan metabolism sequencing revealed a positive correlation between CD73 expression and tryptophan metabolism. In vitro, CD73 overexpression reduced ERK/c-Jun dephosphorylation by decreasing binding to INPPL1, leading to upregulation of IDO1 and TDO2 expression. Co-culture experiments showed weakened CD8 + T cell proliferation in the CD73OE group compared to controls, while inhibition of IDO1/TDO2 significantly enhanced CD8 + T cell proliferation, a result reversed in the knockdown group. The therapeutic effect of an IDO1/TDO2 inhibitor was assessed in an A20 mouse model, where both the AT-0174-treated vector and CD73 OE groups showed decreased tumor growth and increased CD8 + T cell and effector CD8 + T cell infiltration compared to the untreated group. Finally, we investigated whether combining CD73 monoclonal antibody with IDO1/TDO2 inhibitors could enhance immune cell infiltration in tumor tissues, and found that the combination treatment increased CD8 + T cell infiltration. Conclusions: Our study uncovers a novel mechanism by which CD73 regulates immune escape in DLBCL through an adenosine-independent pathway. CD73 overexpression upregulates IDO1 and TDO2 levels by inhibiting ERK/c-Jun dephosphorylation via reduced binding to INPPL1. These findings offer new insights for combination therapies targeting CD73 in DLBCL patients with high CD73 expression.