High expression of ID1 in monocytes is strongly associated with phenotypic and functional MDSC markers in advanced melanoma

A total of 24 advanced stage melanoma patients undergoing surgical removal of resectable metastatic lesions were included. Blood samples of the participants were collected prior to surgery of the melanoma lesions and after a median of 35 days post-surgery (range 14–119 days). The median age of patients at the time of surgery was 63 years (range 44–87 years). An overview of additional patient characteristics is shown in Table 1. Patients did not receive systemic therapy prior to or during the period of sample collection. Samples were analyzed using flow cytometry, in which monocytic MDSC were defined as CD33⁺CD11b⁺CD14⁺HLA-DR^low (see Supplementary Fig. 1 for a full gating strategy).

Patient-derived peripheral blood samples were acquired via the Oncology Department of the Karolinska University Hospital. Blood samples from healthy donors were obtained from the University lab at the Karolinska University Hospital. PBMC were extracted from peripheral blood samples via Ficoll density gradient centrifugation (Ficoll-Paque plus, GE Healthcare Life Science). Patient-derived and healthy donor PBMC were cryopreserved in fetal bovine serum (FBS) with 10% DMSO. PBMC were thawed for analyses by flow cytometry or DC maturation at a later time point.

Isolation of monocytes from fresh or thawed human PBMC was performed using magnetic activated cell sorting (MACS) according to the manufacturer’s instructions (Miltenyi Biotec Cat. No. 130-024-210). Monocytes were isolated using CD14 + microbeads (Miltenyi Biotec Cat. No. 130-050-201) and resuspended in IMDM 10% human AB serum.

Per well 1 × 10⁶ isolated monocytes where plated in 12-well plates (TPP) in 1 ml IMDM with 10% human AB serum (Karolinska University Hospital). Differentiation of monocytes to immature DC (iDC) was done using a fast protocol, in which iDC formation was established by 48 h of culture in the presence of 100 ng/ml GM-CSF (Peprotech) and 20 ng/ml IL-4 (Peprotech). Mature DC (maDC) were created by incubating iDC for an additional 18 h with one of the three following cocktails. The first was the gold standard [26]: 20 ng/ml tumor necrosis factor-α (TNF-α; Peprotech), 10 ng/ml interleukin-1β (IL-1β; CellGenix), 1000 U/ml interleukin-6 (IL-6; CellGenix), and 10 ng/ml PGE2 (SIGMA). Second, the alpha-type 1 polarizing cocktail [27]: 50 ng/ml TNF-α, 25 ng/ml IL1b, 3000 U/ml IFN-α (R&D Systems), 100 U/ml IFN-γ (Imukin^®, Boehringer Ingelheim), and 250 ng/ml polyinosinic:polycytidylic acid (poly I:C, Sigma-Aldrich). Finally, the COMBIG CCK Cocktail [28] was used: 10 ng/ml LPS (Sigma-Aldrich), 20 μg/ml Hiltonol (OncoVir), 2.5 ug/ml R848 (VacciGrade™, InvivoGen), and 1000 U/ml IFN-γ. Readout was performed by flow cytometry.

Single-cell solutions were stained with fluorescent-activated cell sorting (FACS) antibodies to measure protein expression levels. 0.2 × 10⁶ cells were plated in 96-well plate (V-bottom) and washed with PBS. Cells were blocked with 1 µl of IvIgG (Privigen, Germany) for 5 min at room temperature. Next, antibodies for staining of surface markers and dead cell marker were added in PBS in a total volume of 20 µl and kept at 4 °C for 30 min. The following extracellular antibodies were used; CD33 PE-CF594 (BD Biosciences), CD86 PE-Cy7 (Biolegend), HLA-DR APC-Cy7 (Biolegend), CD80 BV421 (Biolegend), Aqua Dead Cell Marker (Life Technologies), CD14 BV570 (Biolegend), CD11b BV605 (Biolegend), and PD-L1 BV786 (BD Biosciences). Samples were washed with FACS buffer (PBS with 1% FBS) and treated for 40 min at room temperature in the dark with 100 µl FoxP3 Fix/Permbuffer (eBioscience). After washing with Perm-wash buffer, samples were stained with intracellular antibodies in Perm-wash buffer in a volume of 20 µl. Staining took place for 40 min at room temperature in the dark. The following intracellular antibodies were used; S100A8/9 FITC (BMA Biomedicals), S100A9 FITC (Biolegend), ID1 PE (LSBio), iNOS PerCP (Santa Cruz Biotechnology), IDO PE-Cy7 (eBioscience), IRF8 APC (eBioscience). Samples were washed three times with Perm-Wash buffer and diluted in 150 µl FACS buffer prior to read-out on the Novocyte Flow cytometer (ACEA Biosciences, Sweden). Analysis was performed using FlowJo (v.10.0.7, Tree Star Inc.). Median or geometrical mean fluorescence intensity (MFI and geoMFI respectively) was used for measurement of protein expression.

As ID1 has been mostly studied in mouse MDSC, we first set out to study in more detail how the expression of known MDSC markers relates to ID1 expression in human monocytic cells [3, 5, 6, 11, 29, 30]. In addition, we investigated to what extent the expression of these markers is affected by a reduction in the tumor burden after surgical removal of melanoma metastases. Therefore, we studied peripheral blood samples collected from 24 stage III and IV melanoma patients. In these samples, we studied ID1 expression in parallel with more established MDSC markers, to evaluate to what extent ID1 can serve as an accurate marker to distinguish HLA-DR^low monocytic MDSC from normal HLA-DR^high monocytes in humans. For a full gating strategy, see Supplementary Fig. 1. Low-to-negative expression of HLA-DR on CD33⁺CD11b⁺CD14⁺ monocytes was defined using the lymphocyte population as an internal control, as the bulk of these cells are negative for HLA-DR. A subpopulation of activated T cells may express HLA-DR at a relatively low level, which was also seen in our samples. We started out by studying levels CD33⁺CD11b⁺CD14⁺ cells for expression of ID1 in relation to markers commonly used for characterization of monocytic MDSC: HLA-DR, iNOS, and S100A8/9. Within the population of CD33⁺CD11b⁺CD14⁺ monocytic cells, we found that the highest expression of ID1 was consistently found in HLA-DR^low cells. At the same time, cells with higher ID1 expression were also more positive for iNOS and S100A8/9 in the same subpopulation of CD33⁺CD11b⁺CD14⁺ cells (Fig. 1a). Interestingly, HLA-DR^low monocytic MDSC displayed a highly significant increase in ID1 expression compared to normal HLA-DR^high monocytes, which coincided with strongly increased levels of S100A8/9 and S100A9 (Fig. 1b). Moreover, iNOS and IDO, two mediators of immunosuppression, were both significantly increased in HLA-DR^low monocytic MDSC, indicative of an immunosuppressive phenotype (Fig. 1b). Finally, HLA-DR^low monocytic MDSC exhibited a strong reduction in IRF8 expression compared to HLA-DR^high monocytes (Fig. 1b). In line with these data, we found that HLA-DR^low cells contained significantly higher frequencies of ID1-positive cells and significantly lower frequencies of IRF8-positive cells (Fig. 1c). No differences could be found for frequencies of cells positive for S100A8/9, however. This is almost certainly caused by the fact that in the large majority of patient samples virtually all monocytes are S100A8/9 positive, whereas S100A8/9 expression levels vary substantially, as illustrated by the S100A8/9 data shown in Fig. 1b.

None of the analyzed markers correlated significantly with the percentages of MDSC in the total cell population, except for HLA-DR (r = − 0.77, p = 0.0001). No inverse correlation between ID1 and IRF8 was present in our samples, even though ID1 was reported to induce MDSC differentiation at least partly by downregulating IRF8 in mice [10].

To get additional evidence for a relation between ID1 expression and expression of more established MDSC markers, we subdivided monocytic cells in S100A8/9^hi and S100A8/9^low cells, while the same was done for S100A9 and iNOS. We found that S100A8/9^high, S100A9^high, and iNOS^high monocytes all expressed significantly higher levels of ID1 compared to, respectively, S100A8/9^low, S100A9^low, and iNOS^low (Fig. 2a). This was further confirmed by the fact that ID1 expression correlated positively with S100A8/9 (r = 0.83, p < 0.0001) and iNOS (r = 0.67, p < 0.0001), but not significantly with S100A9 (r = 0.36, p = 0.0640), as shown in Fig. 2b.

When comparing patient samples before and after surgery, the resection of tumor tissue had no apparent effect on percentages of circulating monocytic MDSC (Supplementary Fig. 2). Interestingly, ID1, S100A8/9, and iNOS expression were significantly decreased after surgery in CD33⁺CD11b⁺CD14 monocytes (Fig. 3). In contrast, an increase in expression after surgery was present for PD-L1 (Fig. 3). With near significance, a trend towards an increase was seen for IRF8 after surgery (Fig. 3). In addition to this, the frequency of cells positive for ID1 and iNOS decreased within the monocytes population after surgery (Supplementary Fig. 3). When analyzing the total frequency of monocytic MDSC positive for the studied markers within all live cells, no changes were observed after surgical removal of melanoma metastases (Supplementary Fig. 4).

After demonstrating that increased ID1 expression in monocytic cells coincides with expression of phenotypic and immunosuppressive markers of monocytic MDSC, we wondered whether the opposite occurs when monocytic cells acquire an immunogenic phenotype. Therefore, we studied ID1 expression during myeloid cell maturation to a fully immunogenic phenotype, using various models for maturation of human monocyte-derived iDC to maDC. To this end, monocytes were isolated from healthy donor PBMC and treated with three different DC maturation cocktails (COMBIG [28], gold standard [26], and α-type 1 polarizing cocktail [27]). Proper maturation of monocyte-derived DC was confirmed by an increase in the percentages of CD80⁺CD86⁺ after exposure of iDC to maturation stimuli (Fig. 4a). During DC maturation, ID1 expression in CD11b⁺CD14⁺ cells significantly decreased, along with reduction in S100A8/9 and S100A9 expression (Fig. 4b). However, we did not see an increase in IRF8 expression after differentiation of monocytes to iDC or further maturation by exposure to any of the cocktails. Unexpectedly, DC matured with the gold standard cocktail actually expressed significantly lower IRF8 levels as compared to cultured monocytes (Fig. 4b).