Blockade of myeloid-derived suppressor cell function by valproic acid enhanced anti-PD-L1 tumor immunotherapy

doi:10.1016/j.bbrc.2019.11.155

Biochemical and Biophysical Research Communications

Volume 522, Issue 3, 12 February 2020, Pages 604-611

https://doi.org/10.1016/j.bbrc.2019.11.155 Get rights and content

Highlights

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VPA-treatment promoted the polarization of bone marrow-derived precursor cells into M-MDSCs.
•
VPA combined with anti-PD-L1 antibody block the immunosuppressive function of MDSCs by downregulating the expression of IL-10, IL-6, and ARG1.
•
Combination therapy of VPA and anti-PD-L1 antibody promoted the activation of IRF1/IRF8 transcriptional axis in MDSCs.
•
The combined treatment enhanced PD-L1 blockade-mediated melanoma regression through the secretion of TNFα.

Abstract

Regardless of the remarkable clinical success of immune checkpoint blockade (ICB) against PD-1/PD-L1 pathway, this approach has encountered drawbacks in most patients due to the activation of tumor immunosuppressive factors such as myeloid-derived suppressor cells (MDSCs). Histone deacetylase (HDAC) inhibitors combat ICB resistance by attenuating the immunosuppressive function of MDSCs and increasing PD-L1 expression on tumor cells. However, whether an HDAC inhibitor - valproic acid (VPA) suppression of MDSCs function could enhance PD-L1 blockade-mediated tumor immunotherapy remains unknown. Here we report that VPA and anti-PD-L1 antibody combined treatment promoted the polarization of bone marrow-derived precursor cells into M-MDSCs. Interestingly, the combination treatment of VPA and anti-PD-L1 antibody activated IRF1/IRF8 transcriptional axis in MDSCs leading to blockade of their immunosuppressive function by downregulating the expression of IL-10, IL-6, and ARG1 while re-activating CD8⁺ T-cells for the production of TNFα to further enhance anti-tumor immunity. These observations provide further rationale for the combination therapy of VPA with anti-PD-L1 antibody in preclinical settings.

Introduction

Immune checkpoint inhibitors (ICIs) targeting programmed cell death 1/programmed cell death 1 ligand 1 (PD-1/PD-L1) axis generate durable clinical responses in a sizeable minority of tumor patients partly through reinvigoration of CD8⁺ T cells [1]. However, several cancer patients have shown overwhelming resistance to immune checkpoint blockades (ICB). The major obstacle to ICB in cancer immunotherapy is activation of different immunosuppressive factors in tumor microenvironment (TME), which inhibit T-cell effector and decrease infiltration of T cells into the tumor tissue.

Myeloid-derived suppressor cells (MDSCs), a major component of pathologically activated cells displaying an exceptional immunosuppressive ability against anti-tumor T-cell response contribute to resistance of ICB. MDSCs are further divided into two subsets: monocytic MDSCs (M-MDSCs) and granulocytic MDSCs (G-MDSCs), respectively [2,3]. Generally, the cell surface markers for MDSCs include Gr1 and CD11b in mice [4,5]. The M-MDSCs are CD11b⁺Ly6C^highLy6G⁻ whereas G-MDSCs are CD11b⁺Ly6C^lowLy6G⁺ [3,4]. More importantly, MDSCs exert its functions through the production of IL-6, IL-10, arginase 1 (ARG1), reactive oxygen species (ROS), and nitric oxide (NO). The differentiation of myeloid cell subsets is coordinated by several transcriptional regulators; interferon regulatory factors (IRF), IRF1 and IRF8 have been identified to be involved in the production of myeloid progenitors [6]. They control various functions; modulation of immune responses, host defense mechanism, cell proliferation, hematopoietic development, and cytokine signaling [7]. IRF8 interacts with other transcription factors such as IRF1 through its association domain to confer immunity against tumor and infectious diseases [8,9]. Blocking immunosuppressive functions of MDSCs leads to markedly enhanced anti-tumor immunity. Interestingly, cancer patients who had a higher level of MDSCs were resistant to anti-PD-L1 therapy. But whether inhibition of MDSCs could relieve resistance to anti-PD-L1 therapy still needs to be investigated.

Histone deacetylases (HDACs) have demonstrated potent anticancer activities, regulation of immune cells function through epigenetic modification of multiple genes [10]. Remarkably, valproic acid (VPA), which is a histone deacetylase inhibitor (HDACi) targeting HDAC class I enzymes (HDAC1, 2 and 3), was reported to have a potential in attenuating the immunosuppressive function of MDSCs [11]. VPA promoted tumor-induced M-MDSCs differentiation into dendritic cells (DCs) and macrophages in-vitro [12]. However, whether VPA suppression of MDSCs function could enhance PD-L1 blockade-mediated tumor immunotherapy remains unknown. In this study, we provide a rationale for combined therapy of VPA and anti-PD-L1 antibody which could be explored in clinical settings.

Section snippets

Mice

Six-to eight-week-old wild-type (WT) C57BL/6 mice were used in this study. Mouse handling and experimental procedures was in accordance with established institute’s guidance and approved protocols from the animal facility committee of Shenzhen Institute of Advanced Technology (SIAT), Chinese Academy of Sciences (CAS).

Cell line

B16F10 or LLC cell line was obtained from CAS cell bank and cultured in DMEM supplemented with 10% FBS, 1% penicillin-streptomycin (PS), 37 °C, 5% CO₂.

Reagent and antibodies

DMEM, RPMI 1640, FBS, PBS and

In vitro

Studies had demonstrated GM-CSF could be used to generate MDSCs in-vitro from bone marrow (BM) precursor cells [13,14]. To investigate the effect of VPA treatment on GM-CSF-induced MDSCs in vitro, we isolated BM cells from mice stimulated with GM-CSF for 3 days, and then treated with either VPA, or anti-PD-L1 antibody, or both VPA and anti-PD-L1 antibody for 24 h, the percentages of MDSCs and its subset were evaluated by flow cytometry. We observed that VPA promoted the differentiation of CD11b⁺

Discussion

This study demonstrated that VPA augments PD-L1 blockade therapy to impair melanoma growth via activation of IRF1/IRF8 transcriptional axis of MDSCs, thus inhibiting MDSCs immunosuppressive function through downregulating IL-10, IL-6, and ARG1 while re-activating CD8⁺ T-cells.

HDACi has been identified as anti-cancer agent by regulating MDSCs generation and function [19]. Class I and II non-selective pan-HDACi (SAHA and TSA) expanded M-MDSCs in in-vitro culture system and in tumor-bearing mice [

Author contributions

Conceptualization, D.Y; Data curation, A.O.A; Formal analysis, A.O.A; Funding acquisition, D.Y and X.W; Investigation, A.O.A; Methodology, W.L; Project administration, X.W; Resources, L.W; Supervision, D.Y and X.W; Validation, M.Z; Visualization, F.O.A; Writing – original draft, A.O.A; Writing – review & editing, D.Y.

Funding

This work was supported by the National Natural Science Foundation of China (Grant 81501356 and Grant 81373112), the Shenzhen Basic Science Research Project (Grants JCYJ20170413153158716 and JCYJ20170818164619194), Nanshan pilot team project (LHTD20160004), Start-up funding (CYZZ20180307154657923), Guangdong Provincial Research Award for Thousand Talents Program Scholars.

Declaration of competing interest

The authors declare no competing financial interests.

Acknowledgements

We appreciate the technical contributions from Lukman. O. Afolabi, Ruiling Liu (Renee) and Jiang Bian during this research.

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The cell pellet was re-suspended in PBS to obtain a single-cell suspension. The single-cell suspension was subjected to mojosort magnetic cell separation according to the manufacturer's procedure and followed by cell surface staining with CD11b and Gr1 monoclonal antibodies for cell sorting on BD FACS Aria III cell sorter (BD Biosciences) as previously described [5]. CD11b+ Gr1+ flow cytometry sorted MDSCs from the spleens of B16F10 tumor-bearing mice treated with or without FATP2 inhibitor (lipofermata) were resuspended in 200μl of PBS and lipid was extracted according to a previously described method [4].
The regulation of myeloid-derived suppressor cells (MDSCs) function is key for effective tumor immunotherapy. Recent lipidomics data revealed that MDSCs accumulate lipid species thereby promote their immunosuppressive activity on T cells. However, genetic manipulation of fatty acid transport protein 2 in mice reduced lipid accumulation in polymorphonuclear MDSCs. Herein we present for the first time lipidome of splenic MDSCs from B16F10 melanoma-bearing mice treated with FATP2 inhibitor – lipofermata compared to the control group. B16F10 were subcutaneously injected into the left flank of wild-type C57BL/6 mice, either lipofermata or vehicle was administered to the mice every day starting from day 7 post-tumor injection for 2 weeks. CD11b⁺Gr1⁺ cells from the spleen referred to as MDSCs were sorted on a flow cytometer machine for lipid extraction. Lipid was extracted using methyl‑tert‑butyl ether as previously described with slight modification, followed by liquid chromatography-mass spectrophotometry lipid profiling using a Q-Exactive instrument coupled with HPLC. The raw scans were identified and quantified with LipidSearch while raw data for various lipid species available on the Mendeley Data repository [1]. The lipid profiles reveal change in lipid species following blockade of FATP2 expression in MDSCs compared to the control. These data were collected in connection to a co-submitted paper [2].
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Phenotypically, these cells are similar to neutrophils and monocytes; however, biochemically and functionally, they are different from the aforementioned cell subsets [5]. In mice, MDSCs are characterized as cells co-expressing CD11b and Gr1 markers, which can be further categorized into two populations: polymorphonuclear (PMN)-MDSCs (CD11b+Ly6G+Ly6Clow) and monocytic (M)-MDSCs (CD11b+Ly6G−Ly6Chigh) [8–10]. Functionally, MDSCs use different mechanisms such as induction of arginase 1 (Arg1), inducible nitric oxide synthase (iNOS), and reactive oxygen species (ROS) production to suppress anti-tumor immune responses [11].
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¹: These authors contributed equally.

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Blockade of myeloid-derived suppressor cell function by valproic acid enhanced anti-PD-L1 tumor immunotherapy

Highlights

Abstract

Introduction

Section snippets

Mice

Cell line

Reagent and antibodies

In vitro

Discussion

Author contributions

Funding

Declaration of competing interest

Acknowledgements

Nature

Curr. Opin. Immunol.

J. Pharmacol. Sci.

Immunity

Biochem. Pharmacol.

Myeloid-derived suppressor cells in the tumor microenvironment: expect the unexpected

J. Clin. Investig.

Subsets of myeloid-derived suppressor cells in tumor-bearing mice

J. Immunol.

The biology of myeloid-derived suppressor cells: the blessing and the curse of morphological and functional heterogeneity

Eur. J. Immunol.

Interferon regulatory factor 8 and the regulation of neutrophil, monocyte, and dendritic cell production

Curr. Opin. Hematol.

Impaired myelopoiesis in mice devoid of interferon regulatory factor 1

Leukemia