Abstract 3165: Stroma-immune landscape in lymphoma: new mechanisms of immunosuppression and therapeutic targeting
Authors: Apollonio Benedetta , Petrov Nedyalko , Spada Filomena , Jarvis Peter , Cozzetto Domenico , Kannambath Shichina , Kuo David , Saqui Mansoor , Amini Rose-Marie , Enblad Gunilla , Charlotte Graham , Benjamin Reuben , Vardi Anna , Phillips Elisabeth , Salisbury Jon , Olson Eric N. , Fox Brian , Hagner Patrick , Gandhi Anita , Jarrett Ruth F. , Herter Sylvia , Bacac Marina , Klaus Christina , Klein Christian , Deutsch Alexander , Ramsay Alan G. 
Published: July 2021

Abstract
Tumor cells engage in bidirectional interactions with stroma and immune cells to promote disease progression and immune evasion. Stroma-specific gene signatures have been associated with outcome in diffuse large B-cell lymphoma (DLBCL), but their immunobiology has been understudied.
To characterize the stromal landscape in lymphoma, we performed high-dimensional imaging mass cytometry analysis of the major stroma subsets and revealed a marked expansion and remodeling of the immuno-specialized fibroblastic reticular cells (FRCs) in human DLBCL biopsies (n=53). The FRC network was similarly remodeled in tumors from the IμBcl6 transgenic model of lymphoma, and aberrant fibroblasts were in close proximity to cancer cells.
Modelling the interactions between murine and patient FRCs and tumor cells, using 2D and 3D cultures, showed that lymphoma drives the acquisition of an inflammatory-like, pro-tumoral (upregulation of fibroblast activating protein-α, FAP) phenotype and associated functional capabilities. Comparative bulk transcriptomic analysis revealed that lymphoma-FRCs undergo transcriptional reprogramming and activate gene pathways associated with inflammatory responses. Moreover, single-cell RNA-seq revealed an expansion of activated FRC clusters expressing B cell supporting genes, while T cell-associated FRCs were contracted. Altered chemokine signaling pathways in DLBCL-FRCs were functionally linked to reduced attraction of T cells and impeded migration along the lymphoma-reticular network. Moreover, lymphoma-FRCs upregulated expression of inhibitory PD-1 ligands that reduced the anti-tumor cytolytic activity of CD8+ T cells, a T cell bispecific antibody (CD20-TCB, glofitamab) and anti-CD19 CAR T cells in our coculture models.
To overcome the immunosuppressive activity of DLBCL-FRCs, we investigated the use of CD20-TCB in combination with stroma-targeting immunocytokine fusion protein drug (FAP-IL2v, RG7461) or costimulatory fusion protein (FAP-4-1BBL, RG7827). Functional cytotoxicity assays using human and murine primary DLBCL patient samples revealed that both stroma-targeting drugs paired effectively with the CD20-TCB to enhance the cytotoxic activity of autologous CD8+ T cells. In addition, the ability of immune-/stroma- targeted combination immunotherapy to trigger anti-tumor activity and CD8+ T cell retention within the FRC-TME was demonstrated using 3D precision-cut lymph node slice-based organotypic cultures of DLBCL and other B cell malignancies.
In conclusion our data reveal that lymphoma cells actively reprogram FRCs that acquire altered immunoregulatory function which prevents effective T cell motility and suppresses the anti-tumor function of cytolytic T cells. Importantly, we demonstrate that combination immunotherapy incorporating fibroblast-targeting fusion proteins could effectively recover anti-tumor T cell activity.
Citation Format: Benedetta Apollonio, Nedyalko Petrov, Filomena Spada, Peter Jarvis, Domenico Cozzetto, Shichina Kannambath, David Kuo, Mansoor Saqui, Rose-Marie Amini, Gunilla Enblad, Graham Charlotte, Reuben Benjamin, Anna Vardi, Elisabeth Phillips, Jon Salisbury, Eric N. Olson, Brian Fox, Patrick Hagner, Anita Gandhi, Ruth F. Jarrett, Sylvia Herter, Marina Bacac, Christina Klaus, Christian Klein, Alexander Deutsch, Alan G. Ramsay. Stroma-immune landscape in lymphoma: new mechanisms of immunosuppression and therapeutic targeting [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 3165.