Introduction: Diffuse large B-cell lymphoma (DLBCL) is the most common type of non-Hodgkin's lymphoma (NHL), which is highly heterogeneous. Approximately 40% of patients still experience refractory or relapsed conditions after receiving the standard RCHOP regimen.In recent years, immunotherapy has completely transformed the treatment landscape for patients with R/R DLBCL. Although CD20/CD3 BsAbs has improved the treatment response rate of relapsed/refractory R/R DLBCL, approximately 60% of patients still show drug resistance to immunotherapy, and the mechanism of resistance is not yet fully understood. This study conducted a longitudinal study on patients treated with CD20/CD3 BsAbs, exploring the drug resistance mechanism of the patients from the perspective of drug-driven changes in the tumor immune microenvironment. Methods: Single cell RNA (scRNA) sequencing samples were collected from R/R DLBCL patients' tumor tissues while the Bulk T cell receptor (TCR) sequencing and flow cytometry samples were collected from peripheral blood (PB). After RNA abstraction, applied 10x Genomics platform for scRNA and TCR sequencing. Results: The results firstly confirmed that all patients treated with glofitamab, the CD20/CD3 BsAbs, experienced T-cell driven. From both scRNA sequencing data and flow cytometry, it's illustrated that in the primary tumor environment and PB, T-cell infiltration proportion in patients with good clinical treatment effects was much higher than that in patients with poor clinical prognosis. Meanwhile, the it's demonstrated the T cell activation effect was significantly enhanced during glofitamab treatment. And Flow cytometry results at different dynamic stages of PB indicated the overall T cell exhaustion of patients decreased during the treatment process. This study included two paired scRNA sequencing data before and after treatment. Currently, all enrolled patients had drug resistance responses, although the results showed the CD8 + effective T cells proportion significantly increased in the primary tumor microenvironment of the patients, the proportion of CD4 + Treg cells and CD8 + exhausted cells also increased significantly. Meanwhile, two different functional groups of CD8 + naive T cells showed distinct changes, the effective group decreased while the exhausted-group increased. This data further suggested the immune system of glofitamab bispecific resistance patients is affected by T cell exhaustion. Despite the effective T cell being driven, it still demonstrated an overall immunosuppressive environment, leading to poor prognosis and disease progression. And the significant changes occurred before and after glofitamab treatment especially on the enriched signaling pathways in T cell activation, including IFN-γ/IL-4 axis and IL-6/IL-23-Th17 signaling pathway. It's suggested drugresistant patients may have a tendency to exhaust activated T cells during CD20/CD3 treatment, promoting an immunosuppressive environment. Furthermore, the significant expansion of shared clones between CD8 + effective T cells and CD8 + exhausted T cells demonstrated by scRNA TCR sequencing results confirmed the important promoting role of T cell exhaustion in patient drug resistance. It indicates the research of glofitamabresistance mechanism may still be concentrated on T-cell exhaustion. Conclusion: In this study, we explored the immune microenvironment of drug-resistant R/R DLBCL patients on CD20/CD3 BsAbs. It's confirmed the T cells driven effect of CD20/CD3 BsAbs. And after that, we described the dynamic T cells profiles, indicated the T cells exhaustion may play the key role in CD20/CD3 BsAbs drug resistance. Most importantly, we suggested the specific T cell subgroups changes during glofitamab treatment, especially CD4 + Treg cell, CD8 + Teff cell, CD8 + T exhausted cell and two types of CD8 + naïve T cell. Through single cell TCRsequencing, we pointed the transformation from CD8 + effective T cell to CD8 + exhausted T cell, indicating the importance of T cell exhaustion in CD20/CD3 BsAbs drug resistance, offering a solid support for the further research on the mechanism of CD20/CD3 BsAbs drug resistance in R/R DLBCL.

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2025
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