Abstract 3333: EMT-6 tumor conditioned Breg cells inhibit NK cell proliferation and cytotoxic activity
Authors: Zhang Yu , Husnain Muhammad , Luo Zhifen , Bayati Mahmood Al , Rosenblatt Joseph D. 
Published: September 2020

Abstract
Purpose: Breg (B regulatory) cells have been implicated in suppression of anti-tumor response in murine and human solid tumors. Prior studies in our laboratory demonstrated that murine EMT-6 breast carcinoma is rejected in B cell deficient mice (BCDM), but not in wild type (WT) mice, due to an enhanced immune response. Tumor infiltrating B cells (TIL-B), and normal splenic B cells co-cultured with EMT-6 (EMT6-B), express LAP/TGF-β, PD-L1, and suppress CD8+ and NK cell proliferation in vitro. Increased CD8+ and NK cell infiltration into the tumor bed is seen in BCDM. BCDM tumor rejection is abrogated by NK depletion. We studied EMT6-B effects on NK cell phenotype, proliferation and cytokine expression in vitro and in vivo.
Experimental design: Normal splenic B cells were co-cultured with mitomycin C treated EMT-6, and Breg phenotype, and effects on NK cell phenotype and proliferation and cytokine function were studied in vitro and in vivo.
Results: EMT6-B but not WT splenic B cells inhibited CFSE labeled CD49b+ NK cell proliferation in response to IL-15 in dose dependent fashion. EMT-6-B suppressed expression of CD226 and NKG2D activating receptors on NK cells, and increased expression of PD-1 and PD-L1 respectively. EMT6-B suppressed the induction of IFNγ+/CD49b+ NK cells following PMA/ION stimulation as assayed by intracellular flow cytometry (10%±1.2 to 2%±0.5) and also suppressed IL-2 induced induction of IFNγ+/CD49b+ NK cells (70%±5 to 20%±7.8). EMT-6 and CT26 colon carcinoma conditioned B cells, but not C166 endothelial cell conditioned B cells suppressed NK degranulation (IFNγ+/CD107a+ NK cells). EMT6-B inhibited YAC-1 cell induced NK cell degranulation, and killing of YAC-1 targets, more effectively than naïve B cells (40%±6.2 to 18%±5.1) (P=0.05). NK suppression was inhibited by anti-PD-L1, and anti-TGF-β antibody. EMT6-B expressed increased FasL compared to splenic B cells, and induced NK apoptosis as measured by 7AAD/Annexin-V staining. Blocking antibody to FasL decreased NK apoptosis, and restored NK cell proliferation.
Infiltrating NK cells in the EMT6 tumor bed demonstrated increased PD-1 compared to splenic and TDLN NK cells, and higher levels of IFN-γ, CD107a, and Granzyme B in BCDM vs. WT mice. EMT6 expressed high levels of CD155 NK inhibitory ligand compared to C166 endothelial cells. The EMT-6 tumor bed contained higher levels of CD19+CD155+ B cells (22% / tumor bed vs 6% / TDLN, vs 9%/ spleen).
BCDM expressed higher levels of CD226+/Ki67+ NK cells in spleen compared to WT splenic NK cells. Antibodies to Fas-L, TGF-beta, and PD-L1 partially restored anti-tumor response in WT mice and decreased tumor growth.
Conclusions: EMT-6 TIL-B or normal B cells conditioned by EMT-6 coculture acquire an NK suppressive phenotype which inhibits NK cell activation, proliferation, degranulation and cytotoxic function. Targeting of Breg-NK interactions may improve anti-tumor immune response.
Citation Format: Yu Zhang, Muhammad Husnain, Zhifen Luo, Mahmood Al Bayati, Joseph D. Rosenblatt. EMT-6 tumor conditioned Breg cells inhibit NK cell proliferation and cytotoxic activity [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3333.