15-, 16- and 17-mer ASOs were selected based on the human NM_001776 or murine NM_001304721 CD39 mRNA (encoded by the ENTPD1 gene). Main criterion for sequence selection was selectivity to avoid undesired off-target effects. LNA modified Gapmers were ordered from Exiqon or Eurogentec and dissolved in H₂O (stock concentration: 1 mM). Antisense oligonucleotides were added to cells without the use of a transfection reagent in vitro and without any delivery system in vivo.

Sequences of ASOs and control oligonucleotides used in the study:Taba

ASO ID	Length	Sequence	Description
A04019H	15	+G* + T* + A*A*G*C*C*C*T*G*A*T* + G* + T* + T	Human specific ASO
A04040H	16	+G* + T* + T*T*G*T*G*T*G*A*G*A*G*C* + T* + T	Human specific ASO, used for in vitro experiments
A04042H	17	+T* + G* + C*C*A*G*A*G*T*G*C*C*T*G* + A* + T* + C	Human specific ASO
A04044H	17	+T* + T* + A*C*G*T*T*C*A*C*T*A*C*C* + T* + T* + C	Human specific ASO
A04045H	17	+C* + A* + C*T*T*A*C*G*T*T*C*A*C*T* + A* + C* + C	Human specific ASO
A04011MR	16	+A* + G* + T*A*A*T*C*C*A*C*C*C*A* + T* + A* + G	Mouse specific ASO, used for in vivo experiments, named: “CD39 ASO” in the text
Control oligo 1	18	+C* + G* + T*T*T*A*G*G*C*T*A*T*G*T*A* + C* + T* + T	Control oligonucleotide, reference: PMID: 26072406, used for in vitro and in vivo experiments
Control oligo 2	17	+T* + C* + T*A*T*C*G*T*G*A*T*G*T*T* + T* + C* + T	Control oligonucleotide, used for in vitro experiments

+ indicates LNA-modified nucleotides and * indicates PTO linkages

RPMI supplemented with antimycotic-antibiotic (100x) and sodium pyruvate (100x) were used at 1% and heat inactivated (30 min at 56 °C) FBS was used at 10% for cell culture experiments (RPMIfs). Cell culture reagents were obtained from GIBCO.

PBMC were derived from Buffy Coats purchased from Blutspendedienst des Bayerischen Roten Kreuzes gemeinnützige GmbH; Hauptverwaltung München; Vertrieb; Herzog-Heinrich-Straße 2; 80336 München or obtained from leukapheresis products.

C57BL/6 and Balb/c mice were bred in-house at University Hospital Basel, Switzerland. In case of unavailability, mice were also obtained from Janvier Labs (France). Animals were housed under specific pathogen-free conditions. All animal experiments were performed in accordance with Swiss federal regulations. Sex-matched littermates at 8–12 weeks of age at start of experiments were used.

Target expression on mRNA level was determined using bDNA assay (QuantiGene SinglePlex Assay Kit 96-Well plate format and QuantiGene Sample Processing Kit for cultured cells, Thermo Fisher Scientific). The following probe sets were used: human ENTPD1 (SA-11803); human HPRT1 (SA-10030); mouse ENTPD1, (SB-13732); mouse HPRT1 (SB-15463). All reagents were purchased from Affymetrix/Thermo Fisher Scientific.

Cells were spun down at 500 g for 5 min, and washed in FACS buffer (1x PBS, 5% FBS) followed by incubation for 25 min at 4 °C in 50 μl FACS buffer per well in 96-well U-bottom plates containing the respective antibodies (anti- human CD8 (clone RPA-T8), anti-human CD4 (clone RPA-T4), anti-human CD39 (clone A1), mouse IgG,κ isotype control and 7-AAD (all from BioLegend). Subsequently, cells were washed twice with FACS buffer and analyzed on a NovoCyte Flow Cytometer (ACEA Biosciences, Inc.).

CD4⁺ and CD8⁺ T cells were separately isolated from PBMCs using MACS (Miltenyi, according to the manufacturer’s instructions). CD4⁺ or CD8⁺ T cells (100,000 per well) were plated on anti-CD3-coated (2 μg/ml; clone OKT3; eBioscience) 96-well U-bottom plates in RPMIfs supplemented with anti-CD28 (2 μg/ml; clone CD28.2; eBioscience) and IL-2 (60 IU/ml; Peprotec) and treated with 5 μM of oligonucleotides for a total treatment time of six days without the use of a transfection reagent. Activation medium and oligonucleotides were replaced after three days. As mock control, cells were cultivated in activation medium without oligonucleotide. On day six after start of treatment, cells were transferred to uncoated 96-well U-bottom plates and cultivated in cell culture medium supplemented with IL-2 (20 IU/ml) in the absence of oligonucleotides. Cells were split 1:2 every third day. hCD39 protein expression was analyzed on day three, six and eleven after removal of oligonucleotides by flow cytometry.

T cells were isolated as described above. Cells were washed twice with PBS, resuspended and adjusted to 2x of the desired final concentration in PBS (pre-warmed to room temperature). A 20 μM solution of cell proliferation dye eFluor™ 450 (eBioscience) in PBS was prepared (pre-warmed to room temperature) and mixed 1:1 with the 2x cell suspension while gently vortexing. Cells were incubated for 10 min at 37 °C in the dark. The labelling reaction was stopped by adding 4–5 volumes of cold complete medium (containing 10% FBS) and cells were incubated on ice for 5 min. Cells were washed 3 times with complete medium before further culturing.

CD8⁺ T cells were isolated from PBMC as described above. 100,000 cells per well were plated on anti-CD3-coated 96 well U-bottom plates in activation medium (as described above) and treated with 5 μM of oligonucleotides for a total treatment time of six days without the use of a transfection reagent. Activation medium and oligonucleotides were replaced after three days. As mock control, cells were cultivated in activation medium without oligonucleotide. Six days after treatment start, cell culture medium was supplemented with ATP (SIGMA-Aldrich) at a concentration of 2 µM. After an incubation time of 30 min, residual ATP concentration in cell culture supernatants or cell-free medium was determined using the ATP Bioluminescence Assay Kit HSII (Roche) according to the manufacturer’s protocol.

CD8⁺ T cells were isolated and labelled with cell proliferation dye as described above. Cells (100,000 per well) were plated on anti-CD3-coated 96 well U-bottom plates in activation medium and treated with oligonucleotides at a concentration of 5 μM for a total treatment time of five days without the use of a transfection reagent. Activation medium and oligonucleotides were replaced after three days. On day three and day four after start of oligonucleotide treatment, 400 μM of ATP or vehicle were added to cells. The following day, hCD39 protein expression, proliferation, and absolute numbers of CD8⁺ T cells were analyzed by flow cytometry (123count eBeads from eBioscience were used to obtain absolute counts). Proliferation index was calculated using the formula: ∑0iNi∑0iNi/2\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$ \raisebox{1ex}{${\sum}_0^i{N}_i$}\!\left/ \!\raisebox{-1ex}{${\sum}_0^i{N}_i/2$}\right. $$\end{document} , whereas i is the generation number and N is the absolute cell count in the respective generation.

C57BL/6 mice were injected subcutaneously into the right flank with 500,000 syngeneic murine MC38 colorectal adenocarcinoma cells (kindly provided by Thomas Wirth, Medizinischen Hochschule Hannover) suspended in phenol red-free DMEM (without additives). Once tumors reached an average volume of 60–80 mm³ (day 12–15), mice were injected intraperitoneally with 200 μl of PBS suspended solutions of CD39 ASO at indicated doses, non-targeting control oligonucleotide (control oligo 1) (100 mg/kg) or left untreated. On day 9 post first compound injection, mice were euthanized and tumors, and in selected cases tumor draining lymph nodes were excised and processed for FACS analyses as outlined below. For tumor growth experiments EMT6 (obtained from ATCC) murine breast cancer cells were injected into the mammary gland of female Balb/c mice. Once tumors reached an average volume of 80 mm³ (Day 8), mice were injected intraperitoneally with 200 μl of PBS suspended solutions of CD39 ASO (20 mg/kg), non-targeting control oligonucleotide (control oligo 1) (20 mg/kg) and/or mouse anti-PD-1 (12.5 mg/kg) (RPM1–14, rat IgG2a, BioXCell) at indicated timepoints. Tumor volume was calculated according to the formula: D /2*d*d, with D and d being the longest and shortest tumor diameter in mm, respectively.

Tumors were harvested from tumor bearing mice and minced using razor blades followed by digestion with accutase (PAA), collagenase IV (Worthington), hyaluronidase (Sigma), and DNAse type IV (Sigma) for 60 min at 37 °C with constant shaking. The cell suspensions were filtered using a cell strainer (70 μm). Lymph node cells were isolated by mashing using the end of a 1 mL syringe. Cells were filtered through a 70 μm nylon mesh. Red blood cells (RBCs) were lysed using RBC lysis buffer (eBioscience). Single cell suspensions derived from tumor and lymph nodes were blocked with rat anti-mouse FcγIII/II receptor (CD16/CD32) blocking antibodies (“Fc-Block”) and stained with live/dead cell-exclusion dye (Zombie UV dye; Biolegend). The cells were then incubated with fluorophore-conjugated antibodies directed against cell surface antigens, washed and resuspended in FACS buffer (PBS + 2%FBS). For intracellular antigens (FoxP3), cells stained with cell surface antibodies were fixed (IC fix, eBioesceince) and permeabilized (Perm buffer; eBioscience) prior to incubation with antibodies directed against intracellular antigens. Cell populations were analyzed on a BD Fortessa. Cells were discriminated using the following combination of cell markers after gating on single cells (discriminated by FSC-A and FSC-H) and excluding non-viable cells (Live/Dead negative). TAMs were denoted by CD45⁺CD11b⁺Ly6C⁻Ly6G⁻F4/80^high. G-MDSC were CD45⁺CD11b⁺Ly6G⁺ and M-MDSC were CD45⁺CD11b⁺Ly6C⁺. CD4⁺/CD8⁺ T cells were CD45⁺CD11b⁻CD3⁺CD4⁺ or CD8⁺. Tregs were FOXP3⁺CD25⁺CD4⁺ T cells. B cells were CD45⁺CD11b⁻CD3⁻CD19⁺. Tumor cells were noted as CD45⁻.

Statistical analysis was performed by GraphPad Prism 7.0 (GraphPad Software). If applicable, results are represented as mean +/− SD. Pairwise comparisons were analyzed using two-tailed student’s t-test and grouped analyses were performed using one-way non-parametric ANOVA with multiple comparison with Tukey’s post hoc test. p-values ≤0.05 (*); p ≤ 0.01 (**); p ≤ 0.001(***); p ≤ 0.0001 (****) were considered significant.

ASOs with specificity for hCD39 were initially screened for target mRNA suppression in HDLM-2 cells without the use of a transfection reagent, a human Hodgkin lymphoma cell line with high endogenous expression of CD39. The most potent candidates were further tested in dose-response experiments. Figure 1a depicts the concentration dependency of target knockdown for the ASOs with the highest activity. The corresponding IC50 values are shown in Additional file 1: Table S1. The most potent hCD39-specific ASO (A04040H, IC50 25.28 nM) was selected for subsequent experiments.Fig1Fig. 1

Selection of hCD39-specific ASO and assessment of target knockdown efficiency. (a) HDLM-2 cells were treated for three days with the indicated concentrations of the respective antisense oligonucleotide without the use of a transfection reagent. hCD39 mRNA expression values were normalized to expression of the housekeeping gene HPRT1. Residual hCD39 mRNA expression relative to untreated cells (set to 100%) is depicted. Depicted is the mean of triplicate wells +/− SD. (b) and (c) Human anti-CD3, anti-CD28, and IL-2 activated CD8⁺ and CD4⁺ T cells were treated with 5 μM of the hCD39-specific ASO A04040H or the control oligo 2 for a total treatment time of six days without the use of a transfection reagent. Thereafter, oligonucleotides were removed and cells were re-plated on plates not coated with anti-CD3. hCD39 protein expression was analyzed by flow cytometry three, six and eleven days after oligonucleotide removal. hCD39 protein expression is depicted as mean fluorescence intensity (MFI) and was calculated by subtracting the MFI of hCD39 by the MFI of unspecific isotype control (b) or as % CD39⁺ cells of alive cells (c). Data is shown as mean of duplicates +/−SD. Asterisks indicate significant differences compared to control oligo treatment within each time point

We assessed the effects of hCD39-specific ASO on the capacity of CD8⁺ T cells and tumor cells (here Burkitt’s lymphoma cells) to degrade extracellular ATP, the substrate of CD39. Cells were treated with A04040H for six days without the use of a transfection reagent. ATP was added to cell culture supernatants or cell-free medium for 30 min after which ATP levels (and CD39 protein expression) were determined. As observed above, treatment with A04040H led to strong suppression of CD39 protein expression compared to control oligo 1 or mock-treated cells (Fig. 2a and Additional file 2: Figure S1A). In cells treated with A04040H, the decreased expression of CD39 correlated with significantly increased levels of extracellular ATP compared to control oligo 1 or mock treatment (Fig. 2b and Additional file 2: Figure S1B). This suggests that the hCD39-specific ASO prevents CD39-mediated degradation of extracellular ATP by targeting CD39 expression.Fig2Fig. 2

Decrease in CD39 expression and extracellular ATP accumulation in human CD8⁺ T cells in presence of hCD39-specific ASO. Human anti-CD3, anti-CD28, and IL-2 activated CD8⁺ T cells were treated with 5 μM of the hCD39 specific ASO A04040H or the control oligo 1 for a total treatment time of six days. hCD39 protein expression was analyzed by flow cytometry. (a) Residual hCD39 expression of oligonucleotide-treated cells relative to mock-treated cells (set to 1). (b) After six days of ASO treatment, 2 μM of ATP were added to cells or cell-free medium. ATP concentration in cell supernatants was determined after 30 min of incubation with ATP and is presented relative to cell-free medium (set to 1). Data is shown as mean of triplicates +/− SD. Asterisks indicate significant differences compared to control oligo treatment

We next investigated the effects of A04040H on CD8⁺ T cell proliferation in the presence or absence of extracellular ATP. A04040H treatment of CD8⁺ T cells potently suppressed CD39 protein expression (Fig. 3a). In the absence of extracellular ATP no significant differences in proliferation index (Fig. 3b) or absolute cell numbers (Fig. 3c) were observed compared to control oligo 2 or mock treatment. As expected, ATP supplementation reduced the proliferation (Fig. 3b) and reduced absolute numbers (Fig. 3c) of control oligo 2 or mock-treated CD8⁺ T cells. This was entirely rescued by treatment with A04040H. We furthermore observed no impact on cell viability upon exposure of A04040H-treated T cells with ATP (Fig. 3d). In contrast, there was a reduction in cell viability (non-significant) when ATP was added to control oligo 2-treated cells and a significant reduction in cell viability when ATP was added to mock-treated cells. In summary, these results reveal that A04040H treatment rescues CD39-induced suppression of T cell proliferation and cell viability, most likely by inhibiting ATP degradation.Fig3Fig. 3

Improved human CD8⁺ T cell proliferation in the presence of hCD39-specific ASO and extracellular ATP. Human CD8⁺ T cells were labelled with cell proliferation dye, activated with anti-CD3, anti-CD28 and IL-2, and treated with 5 μM of the antisense oligonucleotide A04040H or the control oligo 2 for a total treatment time of five days. Subsequently, 400 μM of ATP was added to cells on day three and day four after start of oligonucleotide-treatment. On day five after start of oligonucleotide treatment, (a) hCD39 protein expression, (b) proliferation index, (c) relative cell numbers of CD8⁺ T cells relative to mock-treated cells without ATP (set as 1) as wells as (d) % living cells were analyzed using flow cytometry. Cells were derived from two different donors, processed on three different dates. Bar graphs depict the mean of three independent experiments run in triplicates +/− SD. Asterisks indicate significant differences between the respective conditions

We next utilized syngeneic tumor models to assess the in vivo effects of CD39 ASOs. Upon subcutaneous injection of MC38 colon adenocarcinoma cells, CD39 protein expression was initially assessed on tumor cells and tumor infiltrating immune populations. Myeloid cells, namely TAMs, as well as T lymphocytes expressed higher levels of CD39 compared to B cells and tumor cells (Fig. 4a). CD4⁺ Tregs as well as PD-1 expressing CD8⁺ and CD4⁺ T cell subsets had higher CD39 expression compared to non-Tregs and PD-1 negative cells, respectively (Fig. 4b). Of note, CD39 expression was higher in intratumoral T cells compared to T cells derived from the tumor draining lymph nodes (TDLN) (Fig. 4b).Fig4Fig. 4

CD39 expression on various murine tumor infiltrating lymphocyte and myeloid populations. (a) CD39 expression on live CD45+ immune cells or CD45- tumor cells from freshly digested MC38 murine tumors (around 100 to 200 mm³) of untreated mice was assessed by flow cytometry. TAMs: CD11b⁺Ly6C⁻Ly6G⁻F4/80⁺; G-MDSC: CD11b⁺Ly6G⁺; M-MDSC: CD11b⁺Ly6C⁺. (b) CD39 expression on Tregs (CD4⁺CD25⁺FoxP3⁺) and non-Tregs (CD4⁺CD25⁻FoxP3⁻) as well as PD-1⁺ or PD-1⁻ CD4 and CD8 T cells was assessed in tumors and tumor draining-lymph nodes (TDLN) of untreated mice. (c)-(e) Mice bearing palpable tumors (50–80 mm³) were injected i.p with the indicated doses of CD39 ASO or with 100 mg/kg of control oligo 1. Day 9 post ASO injection tumors were digested and CD39 expression on tumor infiltrating Tregs/non-Tregs (c), TAMs (d) and CD8 (e) was assessed by flow cytometry. Data is represented as fold-change in CD39 MFI compared to control oligo. In all cases each data point represents a mouse. Pooled data from two to three independent repeats. Error bars indicate SD. Asterisks indicate significant differences compared to control oligo 1 group

Tumor infiltrating T cell populations were analyzed by flow cytometry after treatment with mCD39-specific or control oligo 1. A dose-dependent reduction in Treg cell numbers (and frequency) was observed in tumors of mice treated with mCD39-specific ASO while non-Treg CD4⁺ T cell numbers remained unchanged (Fig. 5a, b). Particularly, we noticed that the decrease in Treg frequency was positively correlated with the extent of CD39 expression (Fig. 5c). CD8⁺ T cell frequency did not change upon treatment with the ASO (Additional file 6: Figure S4A). As a consequence of altered Treg cell frequency, the CD8⁺ T cell to Treg ratio was substantially higher in the tumors of mCD39 ASO treated mice (Fig. 5d). This indicates a potential skewing from an immunosuppressive to a pro-inflammatory tumor microenvironment. Accordingly, CD8⁺ T cells in the mCD39-specific ASO treated tumors expressed higher levels of PD-1 (Fig. 5e) and CD25 (Additional file 6: Figure S4B), which may likely reflect increased T cell activation. Compared to the control oligo 1 group, the majority of CD8⁺ T cells in mCD39-specific ASO group were PD-1 positive (40% vs. 96%, Fig. 5 e, middle).Fig5Fig. 5

Changes in tumor infiltrating T cell frequencies upon mCD39-specific ASO treatment. Mice bearing homogenous MC38 tumors (between 50 and 80 mm³) were injected with CD39 ASO at indicated concentrations, or control oligo 1 (100 mg/kg) on days 1–5. On day 8 or 9 tumors were digested to isolate tumor infiltrating immune cells. Frequency of tumor infiltrating Tregs (live CD4⁺FoxP3⁺CD25⁺) (a) and non-Tregs (CD4⁺FoxP3⁻CD25⁻) (b) is indicated as percent of live CD45⁺ cells. (c) CD39 expression on Tregs is depicted against Treg frequency at the respective CD39 ASO doses. (d) Frequency of tumor infiltrating CD8⁺ cells was assessed in tumors of mice treated as per (a) and ratio of CD8⁺ cells to Tregs is depicted. (e) PD-1 expression (MFI) on tumor infiltrating CD8⁺ cells of CD39 ASO and control oligo 1 -treated mice was assessed by flow cytometry. Representative dot plots (middle) and overlapping histograms (right) depict the increase in PD-1 expression on CD8⁺ cells of CD39 ASO treated tumors compared to control oligo 1. In all cases each data point represents a mouse. Pooled data from two to three independent repeats. Error bars indicate SD. Asterisks indicate significant differences compared to control oligo 1 group

Targeting PD-1 on T cells represents a potential strategy to reinvigorate T cell function and offers a rational combination approach. We therefore tested the therapeutic efficacy of systemic CD39 ASO treatment in combination with anti-PD-1 antibody. To this end, we employed an orthotopic breast cancer tumor model by injecting EMT6 cells into the mammary gland (Fig. 6a). While treatment with mCD39-specific ASO resulted in a non-significant reduction in tumor size, combined treatment of mCD39-specific ASO and anti-PD-1 antibody led to significant reduction in tumor burden compared to vehicle control or mCD39-specific ASO monotherapy (Fig. 6b, c); one out of 12 mice rejected the tumor. These data provide a clinically relevant proof-of-principle for the therapeutic utility of CD39 targeting ASO which can be effectively combined with antibodies targeting PD-1.Fig6Fig. 6

Therapeutic efficacy of CD39 ASO in combination with anti-PD-1 antibody. (a) Mice were injected intramammary with 250,000 EMT6 murine breast cancer cells. Once tumors reached 50–80 mm³ (Day 8) mice were injected with CD39 ASO, control oligo 1 and/or anti-PD-1 mAb (all i.p.) on indicated days. Mice were treated with CD39 ASO on five days in the first week and on two days in the second week. Tumor growth is depicted as spider plots (b) and as bar graph indicating cumulative tumor volume on day 25 post cell injection (c). Also included in (c) are euthanized mice reaching termination criteria (tumor volume above 1200 mm³) before day 25. In all cases each data point represents a mouse. Pooled data from two independent repeats. Error bars indicate SD. Asterisks indicate significant differences compared to control oligo 1 group