Histamine targets myeloid-derived suppressor cells and improves the anti-tumor efficacy of PD-1/PD-L1 checkpoint blockade

Abstract

Myeloid-derived suppressor cells (MDSCs) are immature monocytes and granulocytes that impede immune-mediated clearance of malignant cells by multiple mechanisms, including the formation of immunosuppressive reactive oxygen species (ROS) via the myeloid cell NADPH oxidase (NOX2). Histamine dihydrochloride (HDC), a NOX2 inhibitor, exerts anti-cancer efficacy in experimental tumor models but the detailed mechanisms are insufficiently understood. To determine effects of HDC on the MDSC compartment we utilized three murine cancer models known to entail accumulation of MDSC, i.e. EL-4 lymphoma, MC-38 colorectal carcinoma, and 4T1 mammary carcinoma. In vivo treatment with HDC delayed EL-4 and 4T1 tumor growth and reduced the ROS formation by intratumoral MDSCs. HDC treatment of EL-4 bearing mice also reduced the accumulation of intratumoral MDSCs and reduced MDSC-induced suppression of T cells ex vivo. Experiments using GR1-depleted and Nox2 knock out mice supported that the anti-tumor efficacy of HDC required presence of NOX2+ GR1+ cells in vivo. In addition, treatment with HDC enhanced the anti-tumor efficacy of programmed cell death receptor 1 (PD-1) and PD-1 ligand checkpoint blockade in EL-4- and MC-38-bearing mice. Immunomodulatory effects of a HDC-containing regimen on MDSCs were further analyzed in a phase IV trial (Re:Mission Trial, ClinicalTrials.gov; NCT01347996) where patients with acute myeloid leukemia received HDC in conjunction with low-dose IL-2 (HDC/IL-2) for relapse prevention. Peripheral CD14+HLA-DR-/low MDSCs (M-MDSCs) were reduced during cycles of HDC/IL-2 therapy and a pronounced reduction of M-MDSCs during HDC/IL-2 treatment heralded favorable clinical outcome. We propose that anti-tumor properties of HDC may comprise the targeting of MDSCs.

Keywords: Checkpoint inhibition; Histamine dihydrochloride; Myeloid-derived suppressor cells; NOX2; PD-1; Reactive oxygen species.

Conflict of interest statement

Conflict of interest

Authors Hanna Grauers Wiktorin, Frida Ewald Sander, Anna Martner, and Kristoffer Hellstrand hold issued or pending patents protecting the use of histamine and/or α-PD-1/α-PD-L1 therapy in cancer treatment. Kristoffer Hellstrand was previously a consultant to the Re:Mission study sponsor (Meda Pharma). Authors Kristoffer Hellstrand and Anna Martner have received honoraria and/or travel grants from the study sponsor. The other authors declare no potential conflicts of interest.

Ethical approval and ethical standards

The clinical phase IV trial (Re:Mission Trial, ClinicalTrials.gov; NCT01347996), was approved by the Regional ethical review board in Gothenburg on June 2, 2009, application number 267-09. All procedures of this study were performed in accordance with the 1964 Declaration of Helsinki and its later amendments or comparable ethical standards. All animal experiments were performed according to the institutional guidelines and approved by the Research Animal Ethics Committee in Gothenburg on April 4, 2014, application number 86-2014.

Informed consent

Informed consent was obtained from all individual participants before enrollment to the phase IV AML Trial (Re:Mission Trial, ClinicalTrials.gov; NCT01347996).

Animal source

C57BL/6J and BALB/c mice were purchased from the Charles River Laboratories (Sulzfeld, Germany). B6.129S6-Cybbtm1Din (Nox2- KO) mice were originally obtained from the Jackson Laboratory (Bar Harbor, ME, USA) and bred in-house.

Cell line authentication

The EL-4 lymphoma cell line and the 4T1 mammary cancer cell line originated from the American Type Culture Collection (ATCC) and were provided by Ingo Schmitz (Otto von Guericke University, Germany) and Göran Landberg (University of Gothenburg, Sweden), respectively. The MC-38 colon carcinoma cell line originated from the Developmental Therapeutics Program Tumor Repository (Frederick National Laboratory, USA) and was provided by Sukanya Raghavan (University of Gothenburg, Sweden). All cell lines were expanded and frozen in aliquots and were cultured for no more than one week after thawing prior to use in in vivo experiments. Authentication by SNP or STR is not currently standardized for murine cell lines.

Figures

Fig. 1

HDC reduces the growth of EL-4 lymphoma and 4T1 mammary carcinoma in mice. Mice were either untreated (Ctrl, solid lines) or treated with HDC (dashed lines) thrice weekly starting 1 day before tumor cell inoculation. a, b Growth of a EL-4 lymphomas and b 4T1 tumors in wild-type mice. c EL-4 growth in wild-type mice depleted of GR1+ cells. d EL-4 tumor growth in Nox2-KO mice. The tumor size was normalized against the mean tumor size of control mice at the end of each experiment, i.e. against untreated WT mice in a, b, untreated GR1-depleted WT mice in c, and untreated Nox2-KO mice in d. Results were analyzed using two-way ANOVA

Fig. 2

HDC reduces the immunosuppressive properties of MDSCs in mice carrying EL-4 and 4T1 tumors. EL-4-bearing mice were euthanized after 2 weeks and 4T1-bearing mice after 3 weeks of tumor growth when the mean tumor size of untreated mice reached approximately 1.5 cm2. a Accumulation of intratumoral and splenic MDSCs in EL-4-bearing mice. Content of MDSCs was examined in control mice (n = 31 for intratumoral MDSCs, n = 19 for splenic MDSCs) and in HDC-treated mice (n = 33 for intratumoral MDSCs, n = 21 for splenic MDSCs). b Counts of splenocytes in tumor-free (naïve) and control or HDC-treated 4T1-bearing mice. Correlation between c intratumoral MDSCs and tumor size in EL-4-bearing mice or d splenocytes and tumor size in 4T1-bearing mice in control (black) and HDC-treated (grey) animals. e Mean d-peptide-induced ROS production from leukocytes recovered from tumors of control (solid line, n = 18) and HDC-treated (HDC, dotted line, n = 17) EL-4-bearing mice. f ROS formation (area under the curve) in response to d-peptide by single cell suspensions from tumors, spleens or splenocyte-derived GR1+ cells isolated from control (tumor n = 18, spleen n = 20, GR1+n = 9) or HDC-treated (tumor n = 17, spleen n = 19, GR1+n = 11) EL-4-bearing mice. g Mean d-peptide-induced ROS production from leukocytes recovered from tumors of control (solid line, n = 15) and HDC-treated (HDC, dotted line, n = 14) 4T1-bearing mice. h ROS formation (area under the curve) in response to d-peptide stimulation by single cell suspensions from tumors or spleens isolated from control (tumor n = 15, spleen n = 15) or HDC-treated (tumor n = 15, spleen n = 15) 4T1-bearing mice. In f, h ROS formation was normalized against the mean ROS formation of tumor-bearing control mice in each experiment. i, j ROS formation in response to d-peptide from GR1+ (solid line, n = 3) and GR1− (dotted line, n = 3) cells isolated from i tumors and j spleens of control EL-4-bearing mice. k, l Proliferation of OT-1 CD8+ T cells was determined after 3 days of culture. k Representative histograms of CellTraceViolet-stained SIINFEKL-stimulated OT-1 CD8+ splenocytes in the absence of GR1+ cells (SIINFEKL, No GR1+) or in the presence of GR1+ cells isolated from spleens of control or HDC-treated EL-4-bearing mice. l Percentage of proliferating CD8+ T cells in the absence of stimuli (n = 3), in response to a control peptide (gp100, n = 3) or in response to an OT-1 specific peptide (SIINFEKL, n = 3). In specified wells, GR1+ cells that had been isolated from control (n = 5) or HDC-treated (n = 6) EL-4-bearing mice or GR1+ cells isolated from tumor-free mice (n = 2) were present at a 1:1 ratio with the SIINFEKL stimulated OT-1 splenocytes during the course of proliferation. Statistical differences were evaluated using Student’s t test or one-way ANOVA. Linear regression was utilized to analyze correlations. *p < 0.05, ***p < 0.001

Fig. 3

HDC targets human MDSCs in vitro and in vivo. a–c Human monocytes were cultured in the absence of stimuli or in the presence of IL-6 and GM-CSF for 5 days to induce MDSC-like cells. a ROS production from cultured monocytes (ctrl, dotted line) and MDSC-like cells (IL-6 + GM-CSF, solid line) in response to stimulation with fMLF. b Expression of HLA-DR on monocytes after 5 days of culture in absence of stimuli (Ctrl) and in presence of IL-6 and GM-CSF (n = 7). c Expression of HLA-DR on monocytes cultured for 5 days with IL-6 and GM-CSF in the absence or presence of 100 µM HDC (n = 7). d–h AML patients in CR received HDC/IL-2 immunotherapy in 3-week cycles. d Expression of H2R and gp91phox on M-MDSCs for a representative patient. e Frequency and f the absolute counts of M-MDSCs before (cycle 1, day 1; C1D1) and after the first treatment cycle (cycle 1, day 21; C1D21) and at the beginning (cycle 3, day 1; C3D1) and end (cycle 3, day 21; C3D21) of the third treatment cycle. g, h Impact of M-MDSC reduction on leukemia-free survival (LFS) in patients undergoing HDC/IL-2 therapy. Patients were dichotomized by g the median reduction of M-MDSC counts during the first treatment cycle (n = 48), and h the median reduction of M-MDSCs from the start of cycle 1 to the end of cycle 3 (n = 36). Results were analyzed by Student’s paired t test or by the log rank test. *p < 0.05, **p < 0.01, ***p < 0.001

Fig. 4

HDC improves the anti-tumor efficacy of α-PD-1/α-PD-L1 immunotherapy. a EL-4 and MC-38 cells were stained for expression of PD-L1 (solid line) or were left unstained (dashed line). Growth of b EL-4 and c MC-38 tumors in control (solid line), α-PD-1/α-PD-L1-treated (dotted line), or HDC/α-PD-1/α-PD-L1-treated (dashed line) mice. In experiments using EL-4 cells, tumor size was normalized against the mean tumor size of control mice at the end of each of four experiments, and results were analyzed using two-way ANOVA. In the MC-38 model, the difference in slope between HDC/α-PD-1/α-PD-L1 and α-PD-1/α-PD-L1 treatment was analyzed by linear mixed models