Targeting Immune Suppressive Microenvironment By Immune Checkpoint Blockade in Multiple Myeloma
Authors: Gorgun Gullu Topal , Cowens Kristen , Paula Steven , Samur Mehmet Kemal , Ohguchi Hiroto , Singh Ahaana , White Randie E , Bianchi Giada , Suzuki Rikio , Kikuchi Shohei , Harada Takeshi , Mimura Naoya , Tai Yu-Tzu , Hideshima Teru , Laubach Jacob P. , Raje Noopur , Munshi Nikhil C. , Richardson Paul G. , Anderson Kenneth C. 
Published: November 2019

Abstract
Bidirectional interaction between MM cells and accessory cells regulates tumor development on the one hand, while transforming the BM microenvironment into a tumor promoting and immune suppressive milieu on the other. Recent developments in targeted therapies have indicated that generation of the most effective therapeutic strategies requires not only targeting tumor or stroma, but also methods to overcome blockade of anti-tumor immune responses. Tumor associated immune suppressor cells such as Treg and myeloid derived suppressor cells (MDSC) can effectively block anti-tumor immune responses, thereby representing an important obstacle for immunotherapy. Co-inhibitory molecules programmed cell death-1 (PD-1) and its ligand (PD-L1) play a fundamental role in tumor immune escape by inhibiting immune effector functions. Since PD-1/PD-L1 signaling promotes tumor growth while inhibiting effector cell mediated anti-tumor immune responses, we here assessed the impact of PD-1/PD-L1 blockade, alone or in combination with lenalidomide (Len), on accessory and immune cells function, as well as tumor cell growth of MM in the BM milieu.
Methods: PD-L1 gene expression was determined in IFM MM data set (n=170). Cell surface expression of PD-L1 in CD138+ MM cells, stroma, and MDSCs; as well as PD-1 expression on effector cells (CD4T, CD8T, NK, NKT and monocytes/macrophages) were determined in the bone marrow (BM) and peripheral blood (PB) of patients with MGUS (n=3), newly diagnosed (ND-MM, n=5) and relapsed/refractory (RR-MM, n=11) compared to healthy donors (HD, n=10). PD-1/PD-L1 signaling was assessed in the autologous cocultures of patient MM cells or MM cell lines with stroma (BMSC) or MDSCs and effector cells, in the presence or absence of PD-1/PD-L1 blockade and Len. Len effect on PD-1 expression on effector cells and PD-L1 expression on tumor, BMSC and MDSCs was determined by flow cytometry analysis. BMSC and MDSC induced MM growth/viability was measured by MTT, 3H-Thy and CFSE flow cytometry analysis. Effector cell-mediated MM cytotoxicity was measured by CFSE/PI flow cytometry. The effect of PD-1/PD-L1 blockade with or without Len on cytokine pattern was determined by intracellular cytokine flow cytometry analysis.
Results: Statistical analysis of IFM MM data demonstrated that the majority of patient MM cells have increased PD-L1 mRNA compared to HD (p=0.0064). Cell surface expression of PD-L1 was also significantly increased in ND-MM cells (median 65%) and even higher in RR-MM cells. Correspondingly, there was a significant increase in PD-1 expression on CD8T and NK cells in ND-MM and RR-MM. Moreover, PD-L1 expression was significantly higher on both mMDSC and nMDSC than APCs in ND-MM and RR-MM. Coculture with BMSC significantly increased expression of PD-L1 on MM cells. PD-1/ PD-L1 blockade overcame BMSC-induced tumor cell growth in both patient MM cells and MM cell lines. Importantly, Len significantly reduced PD-L1 expression on MM cells; and combined blockade of PD-1/PD-L1 with Len further decreased BMSC-induced MM growth. Immunomodulatory effects of PD-1/PD-L1 blockade were also evaluated in autologous cocultures of immune effector cells with MM cells. Even though there was no change in effector cell proliferation, PD-1/PD-L1 blockade significantly induced cytotoxic activity of autologous T cells, NK cells, and macrophages cultured with MM cells; and Len further enhanced effector cell-mediated cytotoxicity. PD-1/PD-L1 blockade induced intracellular expression of cytotoxic cytokines IFNg and Granzyme B (Gzm B) in CD4T cells, CD8T cells, NK cells and Macrophages. Furthermore, MDSC-mediated MM growth was significantly decreased by PD-1/ PD-L1 blockade. Finally, PD-1/PD-L1 blockade induced intracellular expression of IFNg and Gzm B in T cells, NK cells and NKT cells cultured with autologous MDSC; and Len further enhanced this effector cell activation.
Conclusion: Our data demonstrated that immune checkpoint signaling plays an important role in providing the tumor promoting, immune suppressive microenvironment in MM. Blockade of PD-1/PD-L1 signaling induces anti-MM immune responses that can be enhanced by Len. Targeting checkpoint signaling using PD-1 and PD-L1 blocking antibodies, particularly in combination with Len, therefore represents a promising novel immune-based therapeutic strategy to both inhibit tumor cell growth and restore host immune function in MM.

Disclosures
Kikuchi: The ITO Foundation for the Promotion of Medical Science: Research Funding. Hideshima:Acetylon Pharmaceuticals: Consultancy. Raje:novartis, Amgen, Celgene, Millenium, Onyx: Consultancy; Eli Lilly, Acetylon: Research Funding. Anderson:Celgene: Consultancy; Onyx: Consultancy; Gilead Sciences: Consultancy; Sanofi-Aventis US: Consultancy; Acetylon: Scientific Founder Other; Oncoprep: Scientific Founder Other.