Spatial Architecture of Immune Microenvironment Orchestrating Anti-Lymphoma Immunity and the Response to Immunochemotherapy in EBV+ Diffused Large B Cell Lymphoma Revealed By Imaging Mass Cytometry and Spatial Transcriptome
Authors: Lin Zhijuan , Yu Xingxing , Hong Yan , Liu Long , Li Zhifeng , Zha Jie , Xu Bing 
Published: December 2024

Epstein Barr virus-positive (EBV+) diffuse large B-cell lymphoma (DLBCL), is an aggressive B-cell lymphoma associated with EBV infection characterized by an adverse clinical outcome. In recent years, studies have shown that the immune microenvironment plays an important role in the occurrence and development of EBV+ DLBCL. However, the immune panorama and spatial structure of EBV+DLBCL and its impact on treatment response remain unclear.

We utilized imaging mass spectrometry (IMC) alongside RNA and protein co-staining techniques to locate precisely the Epstein-Barr virus-encoded RNA-positive (EBER+) tumor cells. This allowed for an in-depth analysis of immune component phenotypes and spatial interactions in 24 cases of EBV+ DLBCL and 21 cases of EBV- DLBCL. Meanwhile, spatial transcriptomic was used to decipher the transcriptional signatures of key immune components. Our findings revealed 10 distinct clusters through IMC and we identified two macrophage subtypes in EBV+ DLBCL: CD163+Mϕs and CD163-Mϕs expressing indoleamine 2,3-dioxygenase (IDO), namely CD163-IDO+Mϕs. Compared with EBV-DLBCL, increased infiltration of both CD163+Mϕs and CD163-IDO+Mϕs was observed in the microenvironment of EBV+DLBCL. Spatial transcriptomics further demonstrated that genes associated with antigen receptor-mediated signaling pathways and immune response-activating cell surface receptor signaling were significantly down-regulated in macrophages. It is noteworthy that the infiltration of regulatory T cells (Treg) was significantly decreased in EBV+DLBCL. In the T cell compartment, EBV+DLBCL patients have more functionally exhausted CD8+ T cells, indicated by low levels of granzyme B and Ki-67, and elevated expression of PD-1 and LAG-3. Spatial transcriptomic analysis also revealed down-regulation in genes related to lymphocyte proliferation and activation pathways. Further interaction analysis showed that in EBV+DLBCL, tumor cells received significantly more protection from neighboring macrophages, while receiving significantly less protection from neighboring Tregs. On the other hand, analysis of the proximity interaction of CD8+ T cells showed that in the EBV+DLBCL group, the number of CD8+ T cells hijacked by CD163+Mϕs and CD163-IDO+Mϕs was significantly increased, while the direct interaction between CD8+ T cells and Tregs significantly reduced, indicating a macrophage-mediated immunosuppressive microenvironment.

EBV nuclear antigen 2 (EBNA2) is an important EBV transcription factor that regulates the transcription of EBV latent genes and promotes the expression of important host cell genes that control cell growth and survival. EBV+ DLBCL patients were divided into EBNA2+ and EBNA2- groups. Further subgroup analysis revealed that infiltration of CD8+ and CD4+ T cells was significantly lower in the EBNA2+ group compared to both EBNA2- group and EBV- DLBCL group. In contrast, there were no significant differences in the proportions of macrophages and Tregs between these groups. Interaction analysis showed that tumor cells in the EBNA2+ group were more likely to be shielded by CD163-IDO+Mϕs, making them more adept at evading attacks from CD8+ and CD4+ T cells. Spatial transcriptomics data revealed downregulation of the nuclear factor-κB (NF-κB) pathway and immune checkpoint-related pathways in the tumor cells of EBNA2+ group, with a significant up-regulation of the mitogen-activated protein kinase (MAPK) pathway. To further explore the role of EBNA2 expression in EBV+DLBCL, we assessed the efficacy of immunochemotherapy in the EBNA2+ and EBNA2- group. It turned out that only 2 of the 5 patients in the EBNA2+ group achieved partial response, with 3 experiencing disease progression at mid-term evaluation. In contrast, among the 16 evaluable patients in the EBNA2-group, only 3 developed progressive disease, 3 achieved partial response, and the rest showed an objective response.

In conclusion, EBV+DLBCL has a more immunosuppressive microenvironment, and EBNA2+ patients have significantly reduced T cell infiltration and are associated with poor prognosis. These findings provide a fundamental basis for the prognostic stratification and therapeutic options for EBV+DLBCL patients.