High Level of IL-16 Secreted By Non-GCB DLBCL Facilitates Tumor Growth Though the Tumor-Promoting and Immunosuppressive Tumor Microenvironment

Introduction: Diffuse Large B-Cell Lymphoma (DLBCL) is a highly heterogeneous non-Hodgkin's lymphoma. Based on cell origin, DLBCL is classified into two main molecular subtypes: the germinal center B-cell-like (GCB) subtype and the activated B-cell-like (ABC) subtype. ABC subtype is more aggressive and associated with a worse prognosis than GCB subtype. Previous studies have shown that cytokines play an important role in the growth of ABC subtypes, and IL-16 was supposed to be highly expressed in ABC-DLBCL, however it has not been explored in depth in DLBCL. In this study, we broadly assessed the expression and role of IL-16 on DLBCL. Method: Serum and tissues samples from patients with DLBCL and whole blood from healthy donors were collected. The characterization of protein expression, mRNA expression, and levels of cytokines in cells and tissues was detected by Western blotting, IHC/IHF, PCR, ELISA, RNA-seq, and Flow cytometry. Cells experiments such as cell proliferation, cell apoptosis, cell cycle, cell migration, and ADPC were detected by CCK-8, Flow cytometry, and Transwells. Gene knockdown or overexpression was conducted by lentiviruses and then verified by Western blotting and ELISA. We established subcutaneous tumors (human DLBCL cells and murine DLBCL cells) in BALB/c mice, BALC/c nude mice, and NOD SCID mice for the tumor size or the therapeutic evaluation. Results: First, we found that IL-16, secreted by the cleavage of active caspase-3 from the lymphoma cells under tumor burden, was highly expressed in ABC-DLBCL compared to GCB-DLBCL. The serum IL-16 levels were positively correlated with unfavorable prognosis in DLBCL patients. The dynamic change of serum IL-16 levels accurately predicted the treatment response in patients with DLBCL who received R-CHOP (Rituximab, Cyclophosphamide, Doxorubicin, Vincristine, and Prednisone) or anti-CD19 Chimeric Antigen Receptor (CAR) T cells. Interestingly, bioactive IL-16 showed rare effect on DLBCL cells in vitro, but significantly promoted their growth in vivo. Mechanically, tumor-derived bioactive IL-16 promoted the migration of monocytes via CD4+ receptor, thereby inducing the enrichment of monocytes-derived tumor-associated macrophages in the tumor microenvironment (TME) of DLBCL, which accelerated the tumor growth primarily by expressing cytokines such as IL-6, inducing angiogenesis, and exerting immunosuppressive effects through the expression of cytokines like IL-10; thus, conferring growth advantage to the ABC- DLBCL. To translate the findings into clinical application, we design a novel treatment strategy to overcome the poor outcomes in ABC-DLBCL patients with high IL-16 expression by combination of high-dose cyclophosphamide (CTX) and the bispecific antibody targeting CD20 and CD47 (IMM0306). IMM0306 effectively reversed the tumor-promoting effects of macrophages in subsets of ABC-DLBCL, while CTX induced IL-16 and increased the infiltration of macrophages in the TME. The increasing effector cells/tumor cells ratio further improved the antitumor effect of IMM0306. The combination of high-dose CTX and IMM0306 was confirmed to significantly inhibit the growth of ABC-DLBCL with high IL-16 expression. A clinical trial is being designed to evaluate the potential for clinical applications. Conclusion: In conclusion, this study provides insight into the expression and role of IL-16 in DLBCL. IL-16 is proposed to be highly expressed in ABC-DLBCL, and serum IL-16 indicate the tumor burden, treatment response, and clinical prognosis of DLBCL patients. IL-16 may be a key driver of the TME of DLBCL, which can confer growth advantage to the ABC-DLBCL. Our data provides a theoretical basis for future clinical treatment. A novel treatment regimen (IMM0306 and CTX) could attenuate the pro-oncogenic roles of IL-16 to prevent ABC-DLBCL progression.