Diffuse Large B-Cell Lymphoma (DLBCL) Tumor Cells Reprogram Lymphatic Fibroblasts into Cancer-Associated Fibroblasts (CAFs) That Contribute to Tumor Microenvironment (TME)-Driven Immune Privilege
Authors: Apollonio Benedetta , Nicholas Nicole S. , Sutton Lesley-Ann , Salisbury Jon , Patten Piers E. , Kassam Shireen , Devereux Stephen , Amini Rose Marie , Rosenquist Richard , Ramsay Alan G. 
Published: October 2019

Abstract
There is a clinical need to identify novel treatments for relapsed/refractory DLBCL. Cancer cells engage in novel associations with stromal and immune cells in the TME that provide crucial contributions to disease progression, immune evasion and therapeutic response. However, these hallmark capabilities have been understudied in DLBCL. Given tumor cell genetic complexity, targeting the TME has become a compelling therapeutic strategy. Immune checkpoint blockade therapy (ICB) (e.g. anti-PD-1), which can activate anti-tumor immunity, has provided a new weapon against cancer and serves as an illustrative example of therapeutically re-educating the TME. Clinical results indicate that only a fraction of DLBCL patients currently respond to ICB. Understanding ill-defined TME-driven immune suppression should help optimise ICB and identify novel therapeutic opportunities. Gene expression studies of DLBCL have identified molecular signatures present in both GCB and ABC subtypes related to the TME that correlated with outcome. The prognostically favorable stromal-1 signature reflects reprogrammed stromal cells, extracellular matrix (ECM) and an active immune response. The less favorable stromal-2 signature indicates elevated angiogenesis and blood vessel density. CAFs promote ECM remodelling and angiogenesis in solid cancers. We hypothesized that CAFs play an important role in the pathogenesis of DLBCL including the regulation of subverted host anti-tumor immunity.
To assess whether DLBCL tumor cells induce a CAF phenotype in previously healthy stromal cells, we established a co-culture system with subsequent imaging of conditioned cells. Primary human lymphatic fibroblasts (HLFs) were co-cultured for 5 days in direct contact with a panel of GCB (SU-DHL4, SU-DHL6, DOHH2) and ABC (OCI-LY10, RIVA, U2932) DLBCL cell lines or healthy control B-cells. Quantitative analysis revealed a strong induction of CAF molecular marker expression including FAPα and α-SMA in all DLBCL-educated stromal cells compared to healthy B-cell exposed fibroblasts (P<.01). DLBCL-educated HLFs exhibited dramatic cytoskeletal changes including increased stress fibres. More significantly, the ability of DLBCL-educated HLFs to contract collagen gels, a measure of their matrix remodelling functional capacity, significantly increased compared to control HLFs (P<.01). We next investigated the potential immunomodulatory capacity of DLBCL-educated CAFs using 2-part functional assays. First, healthy T cells were co-cultured (24 h) with either DLBCL-educated HLFs or control HLFs. Second, these T cells were purified and used in subsequent immunologic assays. Exposure to DLBCL-educated HLFs resulted in significant impairment of proliferation of CD4+ and CD8+ T cells in response to anti-CD3/-CD28 (P<.01). The ability of T cells to recognize target tumor cells requires formation of the immunological synapse. We utilized the immune synapse bioassay to examine CD8+ T cell interactions with DLBCL tumor cells. We show that prior co-culture with DLBCL-educated HLFs significantly decreased the formation and strength of CD8+ T cell F-actin immune synapses compared with control HLF co-culture (P<.01). Flow cytometric analysis of FAP+ CAFs revealed markedly increased surface expression of the immune checkpoint ligand PD-L1. The up-regulation of PD-L1 led to the pre-treatment of DLBCL-educated HLFs with an anti-PD-L1 blocking antibody that increased T cell synapse activity. Current experiments are investigating this TME checkpoint axes using primary patient DLBCL tumor cells and T cells. IHC/IF image analysis revealed that PD-L1+ stromal cells reside in the DLBCL TME (archival biopsies, n=20). TME biopsies showed increased expression of α-SMA and FAPα in both GCB and ABC subtypes compared to reactive lymph node samples. CAFs were interspersed within the TME and in close proximity to CD20+ DLBCL tumor cells.
In conclusion, our results establish the ability of DLBCL tumor cells to reprogram HLFs into CAFs that acquire functional capabilities to modulate the TME. Notably, activated CAFs show a compensatory inhibitory response by up-regulating PD-L1 expression that may represent an important TME-driven immunosuppressive mechanism. We believe this data contributes to the understanding of the biology that underlies stromal signatures in the DLBCL TME, in particular the contribution of CAFs to immune privilege.

Disclosures
Ramsay: Celgene: Research Funding; MedImmune: Research Funding.