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Supplemental Figure Legends
Supplemental Figure 1. MIF-inhibitor treatment does not alter the viability of melanoma MDSCs or
A375 melanoma cell line and melanoma patient-derived MDSCs show increased levels of reactive
oxygen species (ROS). (A) Representative histograms showing the percentages of propidium iodide+ (PI+)
cells in melanoma patient-derived MDSCs that were treated for 24 hours with either 0.1% DMSO
(MDSC) or 4-IPP ( MDSC+4-IPP; 50 M). (B) Representative histograms showing the percentages of PI+
cells in A375 cells isolated from A375:monocyte co-cultures that were either untreated (A375) or
treated with 4-IPP (A375+4-IPP; 100 M, day 0 and 50 M, day 2). (C) Representative histogram showing
DCF-detectable ROS levels in CD14+ cells in PBMCs obtained from late stage melanoma patients and
normal donors.
Supplemental Figure 2. Characterization of myeloid cell populations in cultured normal monocytes
and in A375:monocyte co-cultures. Flow cytometry evaluation of expression of CD45, CD11b, CD14,
HLA-DR and CD33 in cultured normal monocytes and in A375:monocyte co-cultures. Representative dot
plots after excluding aggregates and dead cells are shown (FSC/SSC plots; top left). Numbers represent
the percentages from the populations gated. Names above FACS plots indicate the population gated
that was analyzed. Markers analyzed are indicated in the axis of each FACS plot. The gating strategy
used to analyze the samples is illustrated.
Supplemental Figure 3. MIF inhibition during melanoma cell line-induced MDSC alters the acquisition
of MDSC phenotype. (A, B) Representative histograms (A) and bar graph (B) of mean fluorescent
intensities (MFI) of indicated markers expressed on healthy donor monocytes (n = 3) cultured for 64
hours in the absence of melanoma cells (monocytes), or in the presence of the A375 cells. A375 cells and
healthy donor monocytes were co-cultured in the absence (A375-MDSC) or presence of 4-IPP (A3751
MDSC+4-IPP; 100 M on day 0 and 50 M on day 2). (C, D) MIF mRNA (C) and protein expression (D)
were analyzed from cultured monocytes and A375-MDSCs. Data from (B) represent the average ± SEM
of three independent experiments and (C) represents the average ± SEM of duplicate samples and
representative of three independent experiments. P values = *, p≤0.05; **, p≤0.005; ***, p≤0.0005.
Supplemental Figure 4. Conditioned media from A375 cells fail to induce myeloid derived suppressive
function in CD14+ monocytes and MIF inhibition in melanoma cell line-educated CD11b+HLA-DR
MDSCs reduces their suppressive function. (A) CD14+ monocytes from a healthy donor were cultured
for 64 hours in A375 melanoma cell line conditioned media in the absence (A375-CM-Mono), or
presence of 4-IPP (A375-CM-Mono+4-IPP; 100 M, day 0 and 50 M, day 2). Indicated monocytes were
then added to CFSE-labeled autologous T cells and anti-CD3/anti-CD28 beads for 4 days. Representative
histograms indicating the percentage of proliferated T cells are shown. (B) CD11b+HLA-DR MDSCs were
isolated from A375:monocyte co-cultures that were either untreated (A375-MDSC) or treated with 4-IPP
(A375-MDSC+4-IPP; 100 M, day 0 and 50 M, day 2). Indicated MDSCs were then added to CFSElabeled autologous T cells and anti-CD3/anti-CD28 beads for 4 days. Representative histograms
indicating the percentage of proliferated T cells are shown.
Supplemental Figure 5. MIF inhibition in melanoma cell line-educated MDSCs reduces their
suppressive function. (A, B) CD11b+ MDSCs were isolated from A375:monocyte co-cultures (A375MDSC). A375-MDSCs were pre-treated with or without 50 M 4-IPP for 24 hours, washed extensively,
and then added to CFSE-labeled autologous T cells and anti-CD3/anti-CD28 beads for 4 days.
Representative histograms (A) and bar graphs (B) showing the percentage of proliferated T cells. Data
represents the average ± SEM of two independent experiments. P values = **, p≤0.005.
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Supplemental Figure 6. MIF deficiency in murine bone marrow-derived MDSCs (BM-MDSC) reduces
their in vitro suppressive activity. (A, B) MDSC marker expression in BM cells from MIF+/+ and MIF-/- mice
that were cultured for 4 days in vitro in the presence of GM-CSF and IL-6 (BM-MDSC). Representative
dot plots (A) and bar graphs (B) showing the percentages of CD11b+ cells expressing GR1 and CD11c
markers in fresh BM cells and in BM-MDSCs from MIF+/+ and MIF-/- mice. Data represents the average ±
SEM of two independent experiments. P values = *, p≤0.05. (C) BM-MDSCs from MIF+/+ and MIF-/- mice
were added to splenocytes obtained from OT II mice and activated with ovalbumin (OVA; 200 g/mL) for
an additional 72 hours. Eighteen hours before harvesting, co-cultures were pulsed with [3H]-thymidine.
Data represents the average ± SEM of triplicate samples from two independent experiments. P values =
*, p≤0.05; **, p≤0.005.
Supplemental Figure 7. MIF deficiency in murine CD11b+GR1+ bone marrow-derived MDSCs (BMMDSC) reduces their in vitro suppressive activity. (A, B) BM cells from MIF+/+ and MIF-/- mice were
cultured for 4 days in vitro with GM-CSF and IL-6 with or without 4-IPP (50 M) added for the last 48
hours of the 4-day culture period. CD11b+GR1+ BM-MDSCs were isolated from both untreated and 4-IPPtreated MIF+/+ and MIF-/- BM cultures. Indicated CD11b+GR1+ BM-MDSCs were then added to splenocytes
obtained from OT II mice and activated with ovalbumin (OVA; 200 g/mL) for an additional 72 hours.
Eighteen hours before harvesting, co-cultures were pulsed with [3H]-thymidine. Data represents the
average ± SEM of triplicate samples from two independent experiments. P values = *, p≤0.05; **,
p≤0.005; ***, p≤0.0005.
Supplemental Figure 8. Melanoma cell line-induced MDSC transciptome reveals a unique gene
expression profile that is largely reversed to normal monocyte levels by MIF inhibition. Affymetrix
microarray analysis of healthy donor monocytes (n = 3) cultured for 64 hours in the absence (cultured
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monocytes), or presence of A375 cells. A375:monocyte co-cultures were either untreated (A375-MDSC)
or treated with 4-IPP (MDSC+4-IPP; 100 M, day 0 and 50 M, day 2). (A, B) Significantly modulated
genes were clustered into a hierarchical representation. Each column represents one replicate, whereas
each row corresponds to the expression of a particular gene across different replicates/experimental
conditions. The color bar at the left corner of the diagram correlates to the degree of gene expression
with the color scheme: up-regulated genes are represented in red, down-regulated genes in green, and
unmodulated genes in black. (C) Fold change and P-value of selected gene products from the indicated
sample groups are shown.
Supplemental Figure 9. Schematic representation of the timeline for in vitro A375-MDSC induction,
A375-MDSC differentiation and different 4-IPP treatment regimens. (A) Normal donor CD14+
monocytes were co-cultured with human A375 melanoma cells for 68 hours (MDSC induction). CD11b+
A375-MDSCs were isolated from the co-cultures by anti-CD11b+ microbead labeling and magnetic
column separation (MDSC isolation). A375-MDSCs were subsequently cultured in cytokine-free culture
medium for 72 hours and analyzed for DC markers (MDSC differentiation). (B) 4-IPP (50 M) was added
during the A375:monocyte MDSC induction phase following which purified A375-MDSCs were cultured
for 72 hours with no other treatments (A375-MDSC+4-IPP). (C) 4-IPP (50 M) was added only after
MDSC induction during the 72 hour differentiation phase (A375-MDSC treated with 4-IPP).
Supplemental Figure 10. MIF-deficient murine bone marrow-derived MDSCs (BM-MDSC) differentiate
into cells with an immunostimulatory DC phenotype and function. (A, B) BM-MDSCs from MIF+/+ and
MIF-/- mice (n = 2) were cultured ex vivo for 72 hours and analyzed for the DC marker expression by flow
cytometry. Representative dot plots (A) and bar graph (B) showing the percentages of CD80, CD83,
CD86, CD11c, and MHC II IA-IE expressing CD11b+ cells in MIF+/+ and MIF-/- BM-MDSCs post 72-hour
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culture period. (C, D) MIF+/+ and MIF-/- BM-MDSCs were cultured ex vivo for 72 hours. Cultured cells were
added to CFSE-labeled CD4+ T cells purified from splenocytes obtained from OT II mice and activated
with ovalbumin (OVA; 200 g/mL) for an additional 5 days. Representative histograms showing the
percentage of proliferated CFSE-labeled CD4+ T cells (C) and bar graph (D) showing the fold-change in
CD4+ T cell proliferation with MIF-/- BM-MDSCs as compared to proliferation with MIF+/+ BM-MDSCs.
Data represents the average ± SEM of two independent experiments. P values = *, p≤0.05; **, p≤0.005.
Supplemental Figure 11. MIF inhibition in melanoma cell line-educated MDSCs induces an
immunostimulatory dendritic cell (DC) function via suppression of PGE2 production. (A, B) Normal
donor CD14+ monocytes were co-cultured with human A375 melanoma cells for 64 hours (MDSC
induction). CD11b+ A375-MDSCs were isolated from the co-cultures by anti-CD11b+ microbead labeling
and magnetic column separation. A375-MDSCs were subsequently cultured in cytokine-free culture
medium for 72 hours in the absence or presence of 4-IPP (50 M) or 4-IPP (50 M) and PGE2 (10 M)
and analyzed for DC function. Indicated MDSCs were then added to autologous CFSE-labeled T cells in
the presence of Tetanus Toxoid (TT; 1.0 g/mL) for 5 days. Representative histograms (A) and bar graphs
(B) showing the percentage of proliferated T cells.
Supplemental Table 1. List of human antibodies used for flow cytometry.
Supplemental Methods:
MDSC Isolation – A375 co-cultured monocytes (both untreated and 4-IPP treated) and control
monocytes cultured without tumor cells were harvested by gently scraping after 64-68 hours of culture
and CD11b+ cells were purified with anti-CD11b+ microbeads and autoMACS separator (Miltenyi Biotec).
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For some functional studies, CD11b+HLA-DR MDSCs were enriched from the untreated and 4-IPPtreated tumor-monocyte co-cultures: first, CD11b+ cells were immunomagnetically enriched by positive
selection from tumor-monocyte co-cultures. Subsequently, HLA-DR+ cells were removed from the
CD11b+ cell fraction with anti-HLA-DR microbeads.
To dissect the effects of 4-IPP on tumor cells from those on MDSCs, CD14+ monocytes were
cultured in conditioned media obtained from the A375 melanoma cell line. To obtain conditioned
media, A375 cells were cultured in complete IMDM medium (supplemented with 10% human AB serum,
2 mM L-glutamine, and penicillin/streptomycin). Conditioned media was collected after 72 hours or
when the cell confluency was above 80%. Residual tumor cells in the media were removed by
centrifugation and filtration through a 0.2 micron filter. Filtered media was then diluted to 60% with
complete IMDM medium and 3.5 mL of this conditioned media was directly added to CD14+ monocyte
cultures in the presence and absence of 4-IPP. After 68 hours, CD11b+ cells were purified from both
untreated and 4-IPP-treated cultures as described above.
Flow Cytometry – For fluorescence-activated cell sorting (FACS) analysis of the frequency and
phenotype of myeloid cells in melanoma patients, peripheral blood mononuclear cells (PBMCs) from
normal donors (ND) and melanoma patients (MEL) were isolated using Ficoll-Paque density gradients
(GE Healthcare Bioscience). PBMCs were resuspended in FACS buffer [2% FBS + PBS] and counted. 0.5
million live cells (by trypan exclusion) were stained with a multi-color antibody (Ab) panel (Panel 1,
shown below). Following the initial FSC/SSC discrimination, the gate was set on Lineage− (LIN−) Viability
Dye− (to remove dead cells) and CD14+ cells. Next, we gated on the subpopulations defined as MDSC:
CD33, CD11b and HLA-DR cells and their combinations. Gates were set based on isotype controls.
Samples were acquired on FACSCanto II (BD Biosciences).
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To assess the frequency and phenotype of MDSCs in melanoma cell line-educated monocytes,
A375-monocyte co-cultures and cultured monocytes were harvested, washed, counted and
resuspended in FACS buffer and counted. 0.5 million cells live cells (by trypan exclusion) were stained
with multi-color Ab panel (Panel 2, shown below). Following the initial FSC/SSC discrimination to exclude
dead cells and aggregates, the gate was set on CD45+ leukocytes to exclude the tumor cells and then on
CD11b+ cells. Next, we gated on the subpopulations defined as MDSC: CD14, CD33 and HLA-DR cells and
their combinations. Gates were set based on isotype controls.
For characterization of other MDSC/DC markers, separate samples were individually stained
with Ab against the marker of interest in combination with either CD11b Ab or HLA-DR Ab in a two-color
surface staining. For these analyses, FSC/SSC gate was first applied to all samples to exclude dead cells
and cell aggregates and propidium iodide (PI) staining was used in some samples to exclude the dead
cells. Gates were set based on isotype controls or unstained controls. Cell-surface stained cells were
analyzed on a FACSCalibur (Becton Dickinson, Franklin Lakes, NJ).
Panel 1:
Antibody Specificity
/Marker Stained
CD11b
CD14
CD33
HLA-DR
CD3
CD19
Fixable Viability Dye eFluor
780
Flurochrome
FITC
PE
PerCP-Cy5.5
APC
APC-Cy7
APC-Cy7
APC-Cy7
Source
Biolegend
Biolegend
Biolegend
Biolegend
Biolegend
Biolegend
eBioscience
Panel 2:
Antibody Specificity
/Marker Stained
CD11b
CD14
Flurochrome
FITC
PE
Source
Biolegend
Biolegend
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CD33
HLA-DR
CD45
PerCP-Cy5.5
APC
APC-Cy7
Biolegend
Biolegend
Biolegend
Microarray Analysis – A375 co-cultured monocytes (both untreated and 4-IPP treated) and control
monocytes cultured without tumor cells were harvested by gently scraping after 64-68 hours of culture
and CD11b+ A375-MDSCs were purified with anti-CD11b+ microbeads and autoMACS separator (Miltenyi
Biotec). Total RNA from cultured monocytes, vehicle-treated and 4-IPP-treated A375-MDSCs were
isolated. The double-stranded cDNA was synthesized and cleaned up using the GeneChip Sample
Cleanup Module (Affymetrix, Santa Clara, CA). cRNA was synthesized, labeled with biotinylated UTP and
CTP by in vitro transcription. The biotin-labeled cRNA was fragmented and hybridized to the GeneChip®
3′ Human Genome 430 2.0 array (Affymetrix), according to the manufacturer's instructions. The
microarray image data were processed with the GeneChip Scanner 3000 7G (Affymetrix) using the
GeneChip Command Console 1.0 (Affymetrix). The scanned images were analyzed, and the signal
intensity for each gene was calculated with the Affymetrix MAS 5.0 algorithm.
ROS detection – The oxidation-sensitive dye DCF-DA was used to measure ROS production by MDSCs. To
assess the levels of oxidative stress in melanoma patient CD14+ cells, freshly isolated PBMCs from
melanoma patients and healthy donors were incubated with 2.5 M DCF-DA for 20 minutes at 37 °C,
washed twice in ice-cold PBS, stained for surface CD14 marker, and the mean fluorescence intensity
(MFI) of intracellularly retained DCF in CD14+ cells was determined by flow cytometry. To test the effects
of MIF inhibition on the production of ROS in MDSCs, purified melanoma patient MDSCs or A375-MDSCs
were treated with or without 4-IPP (50 M) for 24 hours and, subsequently, incubated at 37 °C in RPMI
in the presence of DCF-DA (2.5 μM) for 30 minutes. For PMA-induced activation, cells were
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simultaneously cultured, along with DCF-DA, with 30 ng/mL of PMA (Sigma-Aldrich) for 30 minutes.
Levels of intracellular DCF were determined by flow cytometry.
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