Program: Oral and Poster Abstracts
Session: 622. Non-Hodgkin Lymphoma: Biology, excluding Therapy: Poster I
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 .
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