KPC-derived PDAC is characterized by infiltration with myeloid cells and a T cell-deprived TME

The KPC-derived T110299 PDAC mouse model shares many pathological features observed in human disease. As such, we investigated the impact of T110299 tumors on myelopoiesis, the TME and its immune cell composition. Tumor cells were implanted into the pancreas of syngeneic C57BL/6 mice and subsequently, immune cell composition in blood, spleen and tumors was monitored within 21 days of tumor development. Tumor engraftment was evident within the first week and progressed rapidly during the following 2 weeks. Tumor growth was paralleled by splenomegaly without any signs of metastasis, indicating influx or proliferation of hematopoietic cells (Fig. 1a-b). Analysis of immune cell composition (Additional file 1: Figure S1) during tumor progression revealed an expansion of myeloid cells in blood, spleen and tumor. The expansion of the myeloid compartment was most pronounced in tumor tissue and identified as CD11b⁺Ly6C^intLy6G⁺ PMN-MDSC and CD11b⁺Ly6C^hiLy6G⁻ M-MDSC, as well as CD11b⁺Ly6C^low/int F4/80⁺ macrophages, as major cell populations (Fig. 1c). In tumors, myeloid cell recruitment (macrophages, PMN-MDSC and M-MDSC) preceded T cell infiltration, with T cells transiently peaking on day 14. On day 21, the immune cell infiltrate was dominated by macrophages and PMN-MDSC. Correlation analysis further revealed a strong correlation between tumor size and PMN-MDSC expansion, both systemically and in tumor tissue (Fig. 1d). Overall, we predominantly observed increased PMN-MDSC populations with increased tumor weight in blood, spleen and tumor, whereas blood CD4⁺ T cells as well as tumor-resident NK cells decreased (Fig. 1e). We also investigated in more detail the role of the growth factor G-CSF, which is produced by KPC-derived PDAC and known to induce proliferation of granulocytic precursor cells in tumor bearing hosts [13]. In our PDAC model, serum levels of G-CSF were increased in tumor-bearing mice, and highly correlated with PMN-MDSC populations in blood and spleen, as well as with tumor weight, suggesting G-CSF to be a major driver for the expansion of PMN-MSC with a strong immunosuppressive phenotype (Fig. 1f).

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Fig. 1

KPC-derived PDAC is characterized by infiltration with myeloid cells and a T cell-deprived tumor microenvironment (TME). T110299 tumors were implanted orthotopically in syngeneic C57BL/6 mice which were sacrificed at days 7, 14 and 21 after tumor induction for analysis of blood, spleen and tumor. a-b Spleen and tumor weights and respective correlation analysis. c Relative frequency of leukocytes in blood, spleen and tumor. d-e Correlation of relative immune cell frequency with tumor weight. f Correlation of serum G-CSF level with PMN-MDSC frequency in blood and spleen as well as correlation of tumor weight with G-CSF level in serum. g-h Surface expression of arginase-1 and PD-L1 on MDSC. i PD-1 expression on T cells in spleens and tumors. j MDSC-like cells from naïve mice as well as MDSC from spleens and tumors of tumor-bearing mice were isolated and co-cultured with CFSE-labeled T cells in increasing effector (E; MDSC) to target (T; T cell) ratios (E:T) of 0.25:1, 0.5:1 and 1:1, in the presence of anti-CD3/anti-CD28 mAb-coated beads. After 72 h CFSE dilution of T cell populations was assessed. a,c,g,h,i Data ± SEM is shown for n = 4–5 mice per group. b,d,f n = 12 mice (e) n = 12 mice / group (c) Statistics for the comparison of day 0 and day 21 (blood and spleen), and day 7 and day 21 (tumor) are shown. (j) Representative graph of three independent experiments, Data± SEM for n = 2 mice per group, unpaired two-sided students t test (*p < 0.05, **p < 0.01, in J compared to tumor-free control)

The inverse correlation of MDSC and T cell infiltration during PDAC progression prompted us to characterize immune suppressive mechanisms of the TME. Within the MDSC compartment, we investigated the expression of known immune suppressive mediators, such as arginase-1 and the checkpoint molecule PD-L1. Arginase-1 levels were comparably low in splenic PMN- and M-MDSC during tumor development, but highly upregulated in tumor-resident MDSC (Fig. 1g). Similar characteristics were also found for PD-L1 expression (Fig. 1h). Moreover, the PD-L1 counterpart, PD-1, was expressed on the vast majority of tumor-resident CD8⁺ and CD4⁺ T cells (Fig. 1i). Subsequently, we assessed the ability of MDSC to inhibit T cell activation, which is the population-defining hallmark of MDSC. We isolated MDSC populations from spleen and tumors to set up a co-culture with anti-CD3/CD28 mAb-activated T cells from tumor-free mice, and used monocytes and granulocytes isolated from spleens of tumor-free mice as controls. Only MDSC from PDAC-bearing mice displayed pronounced suppressive effects on CD8⁺ as well as CD4⁺ T cell proliferation. Whilst PMN-MDSC turned out to be more suppressive than M-MDSC, overall MDSC populations isolated from tumors exhibited the most pronounced suppressive capacity (Fig. 1j). Together, the data show that KPC-derived PDAC develop typical features of a suppressive TME characterized by pathologically activated myeloid cells with high suppressive capacity.

Poly(I:C)_c reduces tumor mass in PDAC concomitant with enhanced T cell activation and reduced suppressive capacity of MDSC

We showed earlier that a systemic therapy with the MDA5 ligand poly(I:C)_c had a positive effect on survival of PDAC-bearing mice, which was dependent on the presence of cytotoxic T cells [27]. Other studies with RLH ligands pointed towards a reduced number or altered function of MDSC in treated animals [24, 28, 40]. To study the effect of RLH activation on MDSC in fully established tumors in more detail, we treated mice with poly(I:C)_c i.v. and analyzed the tumors 21 days after tumor induction. Treatment resulted in a 50% reduction in tumor mass (Fig. 2a). Downregulation of MHC class I is a common mechanism of tumors to evade the immune system. We could previously show that stimulation of pancreatic cancer cells in vitro with RLH ligands induces the up-regulation of MHC-I as well as CD95 (Fas), resulting in more effective tumor cell killing by cytotoxic T cells [26]. In line with these in vitro findings, poly(I:C)_c led to a profound upregulation of MHC-I molecules on tumor cells in vivo (Fig. 2a). RLH ligands are strong inducers of type I IFN, which in turn mounts a robust immune response connecting innate with adaptive immunity. As such, RLH treatment resulted in high levels of CXCL10 and IFN-β, which were accompanied by T_h1-supporting IL-12p70 and IFN-y as well as IL-28, an important type III IFN further supporting CTL-mediated cytotoxicity (Fig. 2b).

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Fig. 2

Poly(I:C)_c reduces tumor mass in PDAC concomitant with enhanced T cell activation and reduced suppressive capacity of MDSC. Mice with orthotopic T110299 tumors were treated with poly(I:C)_c twice prior to sacrifice at day 21 after tumor induction. a Tumor weights, tumor cell MHC-I expression and (b) serum cytokine levels. c Frequencies of MDSC populations in spleen and tumor of untreated and poly(I:C)_c-treated mice. d Surface expression profiles of PD-L1 on MDSC subsets. e Frequencies of T cell populations in spleen and tumor of untreated and poly(I:C)_c-treated mice. f-g CD69 and PD-1 surface expression of splenic and tumor-resident T cells. h Representative data of IFN-γ secretion in MDSC / T cell co-cultures, at a ratio of 1:1, following anti-CD3/anti-CD28 mAb-coated beads stimulation for 72 h. i Splenic T cells and MDSC from spleens and tumors of untreated or poly(I:C)_c-treated tumor-bearing mice were isolated and co-cultured with CFSE-labeled T cells in increasing effector (E; MDSC) to target (T; T cell) ratios (E:T) of 0.25:1, 0.5:1 and 1:1 in the presence of anti-CD3/anti-CD28 mAb-coated beads. After 72 h CFSE dilution of CD4⁺ and CD8⁺ T cells was assessed. a-f Data ± SEM is shown for n = 5 to 8 mice per group. g-h Representative graph of three independent experiments. Data± SEM for n = 2 mice per group,unpaired two-sided students t test (*p < 0.05; **p < 0.01)

We observed a relative reduction of PMN-MDSC, whereas M-MDSC frequency increased in both spleen and tumor (Fig. 2c). Analysis of MDSC surface marker expression revealed a strong therapy-mediated PD-L1 induction in spleen. Similar to our earlier observations in treatment-naïve mice, we found high basal PD-L1 expression by MDSC within the tumor tissue, which was not further increased by immunotherapy (Fig. 2d). T cell frequencies in spleens were unaltered; however, increased infiltration of CD8⁺ T cells was detected within the tumor tissue, which is in line with our previous observations (Fig. 2e). Both splenic and tumor-infiltrating T cells upregulated expression of the early activation marker CD69 in response to poly(I:C)_c, whereas PD-1 expression was unaffected (Fig. 2f-g). We then assessed whether poly(I:C)_c treatment affects the suppressive capacity of MDSC. We isolated MDSC populations from spleen and tumor of untreated and treated PDAC-bearing mice and studied their influence on T cell proliferation. In order to assess overall immunosuppressive effects of MDSC T cell response, we measured IFN-γ secretion as key cytokine of T cell activation in MDSC co-cultures. As expected, at an effector (MDSC) to target (T cell) ratio of 1:1, IFN-γ secretion was strongly suppressed by MDSC (Fig. 2h); however, this was - at least in parts - rescued in co-cultures with MDSC from mice previously treated with poly(I:C)_c. As observed before, tumor-derived MDSC were more suppressive compared to their splenic counterparts, with the highest suppression seen in PMN-MDSC co-cultures, for both CD8⁺ and CD4⁺ T cells. Suppressive function of MDSC populations from poly(I:C)_c-treated animals was reduced for both, PMN-MDSC and M-MDSC (Fig. 2i). These findings are indicative of a functional in vivo reprogramming of MDSC in poly(I:C)_c-treated mice.

Analysis of splenic and tumor-resident B and NK cells showed a slight increase in splenic B cell numbers; both cell populations upregulated CD69 expression upon therapy (Additional file 1: Figure S2A-B). Poly(I:C)_c treatment increased both the intratumoral frequency of migratory cross-presenting conventional DC 1 (cDC1) as well as their activation measured by CD40 expression. In addition, the co-stimulatory molecule CD86 was upregulated in both CD11c⁺MHC-II^hiCD26⁺XCR1⁺CD172a⁻ cDC1 and CD11c⁺MHC-II^hiCD26⁺CD172a⁺XCR1⁻ cDC2 in the tumor-draining lymph node (Additional file 1: Figure S2C-D). Interestingly, the relative frequency of macrophages/TAM was significantly reduced in treated animals, in both spleen and tumor. Moreover, macrophages/TAM showed an activated phenotype with enhanced MHC-I expression (Additional file 1: Figure S2E-G). Further analyses revealed that the frequency of M2-like CD204⁺CD206⁺ macrophages, known to highly correlate with poor disease outcome in patients with various cancer types [41–43], was decreased in tumors (Additional file 1: Figure S2E-F). TAM showed a strong T cell suppressive phenotype, which was – in contrast to MDSC – not reversed upon poly(I:C)_c treatment (Additional file 1: Figure S2H).

Transcriptomic profiling reveals a therapy-induced reprogramming of MDSC

To better understand the mechanisms by which MDSC undergo phenotypical changes upon systemic immunotherapy, we performed a whole transcriptome analysis of PMN- and M-MDSC populations from spleens and tumors. Mice with orthotopic PDAC were treated on day 18 and 20 after tumor implantation with poly(I:C)_c or were left untreated. On day 21, MDSC were sorted for high purity (Additional file 1: Figure S3A-B), followed by RNA extraction and next-generation sequencing. An unbiased principle component analysis (PCA), using the ~ 14.000 most expressed genes, was performed. For both PMN- and M-MDSC, the replicates of each condition clustered closely, confirming the high quality of the data. PCA revealed that PC1 distinguishes samples based on the compartment they were isolated from (PMN-MDSC: 44.3%; M-MDSC: 25.5%), and PC2 described the changes that were induced by poly(I:C)_c treatment (PMN-MDSC: 10.5%; M-MDSC: 15.6%) (Fig. 3a). The 1000 genes contributing most to PC2 in PMN- and M-MDSC show a similar regulation in both spleen and tumor (Fig. 3b).

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Fig. 3

Poly(I:C)_c triggers transcriptional reprogramming of MDSC. Mice with orthotopic T110299 tumors were treated twice with poly(I:C)_c prior to sacrifice as described before. RNA of MDSC populations was isolated for whole transcriptome analysis. a Principal component analysis (PCA) of transcriptome of splenic or tumor-derived MDSC with and without poly(I:C)_c treatment. b Heatmap of gene expression values (colors indicate row z-scores) for the 1.000 genes contributing most to principle component 2 (PC2). c DAVID analysis for enriched gene ontology biological processes (GO:BP) terms from differentially expressed genes (adjusted p < 0.001, ≥ 2-fold change) upon poly(I:C)_c treatment from splenic MDSC. d Gene set enrichment analysis (GSEA) of differentially expressed genes upon poly(I:C)_c treatment compared to published gene sets describing PMN-MDSC vs. neutrophils (GSE24102) and macrophage polarization (GSE5099). Data shown for n = 3 to 4 mice per group

The treatment-induced transcriptomic changes were analyzed by a differential gene expression analysis (adjusted p < 0.001, ≥ 2-fold change) in PMN-MDSC (spleen: 420; tumor: 180; shared: 100) and M-MDSC (spleen: 584; tumor: 210; shared: 113) (Additional file 1: Figure S3C). Functional annotation analysis using the Database for Annotation, Visualization and Integrated Discovery (DAVID) was done with differentially expressed genes from spleen. Genes were found to be significantly enriched in gene ontology biological process (GO:BP) clusters related to immune system processes, virus and IFN response-related pathways, and antigen presentation-related genes (Fig. 3c and Additional file 1: Figure S4). Most importantly, gene set enrichment analysis of splenic differentially expressed genes revealed an enrichment of neutrophil-associated gene signature for PMN-MDSC and enrichment of M1-associated genes for M-MDSC after poly(I:C)_c therapy, suggesting the phenotypic reprogramming of MDSC (Fig. 3d).

MDSC of treated mice do not acquire professional antigen presenting cell function

One of the significantly enriched gene clusters was associated with MHC class-I antigen presentation. In both PMN- and M-MDSC, essential components of the MHC-I-dependent antigen processing and presentation machinery, including the immunoproteasome, the peptide transporter TAP and the MHC-I complex, were up-regulated following poly(I:C)_c therapy (Fig. 4a-b). Flow cytometric analysis revealed a therapy-induced upregulation of MHC-I expression for PMN-MDSC in spleen and tumor, and for M-MDSC in spleen only (Fig. 4c). Moreover, upregulation of the costimulatory molecule CD86 was observed in a subset of splenic PMN-MDSC and the majority of M-MDSC. Tumor-resident M-MDSC already expressed high levels of CD86 and remained unaltered upon therapy (Fig. 4d).

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Fig. 4

MDSC of treated mice do not acquire professional antigen presenting cell function. a-b Schematic representation of differential gene expression upon poly(I:C)_c treatment annotated in the KEGG pathway antigen processing and presentation of PMN- and M-MDSC. c-g Mice with orthotopic ovalbumin expressing PDAC (T112099-OVA) were treated with poly(I:C)_c twice prior to sacrifice at day 21 after tumor implantation. c-d Surface expression of MHC-I and CD86⁺ of MDSC populations at baseline and upon poly(I:C)_c treatment. e-g MDSC from (e) tumor and (f-g) spleen of untreated or poly(I:C)_c-treated tumor-bearing mice were isolated. Splenic MDSC were either treated with OVA protein (f) or SIINFEKL peptide (g). Subsequently MDSC were co-cultured with CFSE-labelled OT-I T cells with an increasing effector (E; MDSC) to target (T; T cell) ratio (E:T) of 0.25:1, 0.5:1 and 1:1 and CFSE dilution of CD8⁺ T cells was assessed following 72 h of co-culture. c-d Data are shown for n = 5 to 6 mice per group. e-g Representative graph of two independent experiments, Data± SEM for n = 2 mice per group (n.d. = not determined; *p < 0.05; **p < 0.01)

To investigate the ability of MDSC to present tumor-associated antigen on MHC-I, ovalbumin (OVA)-expressing T110299 tumors (T110299-OVA) were used as model. 18 and 20 days after tumor induction, mice were treated with poly(I:C)_c or left untreated and MDSC from both tumor and spleen were isolated. Tumor-derived PMN- and M-MDSC were unable to induce antigen-dependent CD8⁺ T cell proliferation, irrespective of treatment (Fig. 4e). In Addition, we evaluated the capability of MDSC to process and cross-present OVA protein ex vivo. Splenic MDSC from T110299-OVA tumor-bearing hosts were incubated overnight with OVA protein and subsequently co-cultured with OT-I T cells for 3 days. Again, no T cell proliferation was detectable (Fig. 4f). To rule out that the lack of functional cross-presentation is due to T cell inhibition by MDSC, the presentation of exogenously added SIINFEKL peptide was assessed. For this, MDSC of T110299-OVA tumor bearing hosts were isolated, pulsed with SIINFEKL peptide and subsequently co-cultured with OT-I T cells. Peptide-loaded MDSC were able to induce a strong OT-I T cell proliferation, with no detectable differences between MDSC of untreated or treated mice (Fig. 4g). Together, these data rule out a function of MDSC as professional antigen presenting cells, which was irrespective of their polarization status.

Therapeutic efficacy and immune activation of MDA5-targeted immunotherapy is mediated by type I IFN signaling

MDA5 activation is known to induce type I IFN and the transcriptomic profile of MDSC in poly(I:C)_c treated mice confirmed a predominant type I IFN response. To further evaluate the role of IFN signaling on MDSC function and tumor control, therapeutic efficacy of poly(I:C)_c treatment was assessed in PDAC-bearing wild-type and IFNAR1-deficient mice.

Tumor weight was significantly decreased in wild-type mice after poly(I:C)_c treatment, whereas no difference was observed in Ifnar1 ^−/− mice, supporting a role of IFN signaling as a prerequisite for anti-tumor efficacy (Fig. 5a). As expected, CXCL10 serum levels of both wild-type and Ifnar1 ^−/− mice were comparable after treatment; however, IL-6 serum levels were significantly decreased in Ifnar1 ^−/− mice (Fig. 5a). Untreated mice had comparable frequencies of MDSC and poly(I:C)_c treatment led to a decrease of PMN-MDSC and an increase of M-MDSC numbers in wild-type mice, but not Ifnar1 ^−/− mice (Fig. 5b). Furthermore, MDSC from IFNAR1-deficient mice failed to upregulate MHC-I and PD-L1 expression upon therapy, indicating a critical role for IFN signaling on MDSC numbers and phenotype upon MDA5-based immunotherapy (Fig. 5c-d). Neither the genotype nor the treatment had an influence on CD4⁺ and CD8⁺ T cell frequencies in spleen and tumor; however, poly(I:C)_c failed to induce CD69 expression in T cells of Ifnar1 ^−/− mice (Fig. 5e-f).

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Fig. 5

Bidirectional control of MDSC suppressive function by type I interferon signaling in PDAC. Wild type and IFNAR1-deficient mice were transplanted with T110299 orthotopic tumors and treated with poly(I:C)_c twice prior to sacrifice at day 21 after tumor induction. a Tumor weights, CXCL10 and IL-6 serum levels in untreated and treated mice. b Splenic MDSC frequency. c-d MHC-I and PD-L1 surface expression on splenic MDSC. e-f T cell frequency and CD69 expression on splenic T cells. g Splenic T cells from untreated C57BL/6 mice and MDSC from spleens of T110299 tumor-bearing wild type or IFNAR1-deficient mice were isolated and T cell suppression was analyzed ex vivo. T cells were co-cultured with an increasing effector (E; MDSC) to target (T; T cell) ratio (E:T) of 0.25:1, 0.5:1 and 1:1 for 72 h in the presence of anti-CD3/anti-CD28 mAb-coated beads. CFSE dilution of CD4⁺ and CD8⁺ T cell populations was assessed. a-f Data ± SEM is shown for n = 4 to 7 mice per group. g Data± SEM for n = 3–5 mice per group, unpaired two-sided students t test(*p < 0.05; **p < 0.01, (g) comparison of untreated wild type and untreated IFNAR1-deficient are depicted with *; #p < 0.05, ##p < 0.01, comparison of untreated wild type and treated wild type are depicted with #)

Our data show that MDA5-based immunotherapy in PDAC-bearing mice led to a reduction of the suppressive function of MDSC populations, concomitant with a dominant IFN signature in their transcriptomic profile. We therefore investigated the role of type I IFN signaling on the suppressive capacity of MDSC in Ifnar1 ^−/− mice. Interestingly, in untreated tumor-bearing hosts the suppressive capacity of MDSC was reduced in Ifnar1 ^−/− mice, as compared to their wild-type controls, pointing towards a role for IFN signaling in early MDSC differentiation into a suppressive phenotype (Fig. 5g). Of note, while poly(I:C)_c treatment reversed the suppressive capacity of MDSC in wild-type mice, the T cell suppressive function of both PMN- and M-MDSC from Ifnar1 ^−/− mice was not significantly changed, arguing for a role of IFN signaling in regulating the suppressive function upon MDA5-based therapy.