Functional characterization of human Cd33+ And Cd11b+ myeloid-derived suppressor cell subsets induced from peripheral blood mononuclear cells co-cultured with a diverse set of human tumor cell lines
© Lechner et al; licensee BioMed Central Ltd. 2011
Received: 31 May 2011
Accepted: 9 June 2011
Published: 9 June 2011
Tumor immune tolerance can derive from the recruitment of suppressor cell populations, including myeloid-derived suppressor cells (MDSC). In cancer patients, MDSC accumulation correlates with increased tumor burden, but the mechanisms of MDSC induction remain poorly understood.
This study examined the ability of human tumor cell lines to induce MDSC from healthy donor PBMC using in vitro co-culture methods. These human MDSC were then characterized for morphology, phenotype, gene expression, and function.
Of over 100 tumor cell lines examined, 45 generated canonical CD33+HLA-DRlowLineage- MDSC, with high frequency of induction by cervical, ovarian, colorectal, renal cell, and head and neck carcinoma cell lines. CD33+ MDSC could be induced by cancer cell lines from all tumor types with the notable exception of those derived from breast cancer (0/9, regardless of hormone and HER2 status). Upon further examination, these and others with infrequent CD33+ MDSC generation were found to induce a second subset characterized as CD11b+CD33lowHLA-DRlowLineage-. Gene and protein expression, antibody neutralization, and cytokine-induction studies determined that the induction of CD33+ MDSC depended upon over-expression of IL-1β, IL-6, TNFα, VEGF, and GM-CSF, while CD11b+ MDSC induction correlated with over-expression of FLT3L and TGFβ. Morphologically, both CD33+ and CD11b+ MDSC subsets appeared as immature myeloid cells and had significantly up-regulated expression of iNOS, NADPH oxidase, and arginase-1 genes. Furthermore, increased expression of transcription factors HIF1α, STAT3, and C/EBPβ distinguished MDSC from normal counterparts.
These studies demonstrate the universal nature of MDSC induction by human solid tumors and characterize two distinct MDSC subsets: CD33+HLA-DRlowHIF1α+/STAT3+ and CD11b+HLA-DRlowC/EBPβ+, which should enable the development of novel diagnostic and therapeutic reagents for cancer immunotherapy.
Keywordsmyeloid-derived suppressor cells tumor immune tolerance human tumor cell lines immunomodulation cytokines hypoxia-inducible factor 1 alpha CAAAT-enhancer binding protein signal transducer and activator of transcription inflammation
Myeloid-derived suppressor cells (MDSC) have recently been recognized as a subset of innate immune cells that can alter adaptive immunity and produce immunosuppression . In mice, MDSC are identified by CD11b+, IL-4Rα+, and GR-1low/int expression, with recognized granulocytic and monocytic subsets [2–6]. Human MDSC are less understood and comprise a heterogeneous population of immature myeloid (CD33+) cells consisting of dendritic cell, macrophage, and granulocyte progenitors that lack lineage maturation markers [2, 5]. MDSC inhibit T cell effector functions through a range of mechanisms, including: arginase 1 (ARG-1)-mediated depletion of L-arginine , inducible nitric oxide synthase (iNOS) and NADPH oxidase (NOX2) production of reactive nitrogen and oxygen species [8, 9], vascular endothelial growth factor (VEGF) over-expression , cysteine depletion , and the expansion of T-regulatory (Treg) cell populations [12, 13]. While rare or absent in healthy individuals, MDSC accumulate in the settings of trauma, severe infection or sepsis, and cancer , possibly as a result of the hypoxia and inflammatory mediators in the tumor microenvironment [14–19]. In cancer patients and experimental tumor models, MDSC are major contributors to tumor immune tolerance and the failure of anti-tumor immunity . Given the multitude of immune modulatory factors produced by tumors, it is indeed quite likely that different subsets of MDSC may be generated in the tumor microenvironment dependent upon the unique profile of factors secreted by the tumor [16, 17, 20]. Preclinical models of human tumor-induced MDSC will significantly advance knowledge of their induction and function as suppressor cells.
In a prior study, we demonstrated that certain cytokines can induce CD33+ MDSC from normal donor peripheral mononuclear cells . As an extension of these studies, we now report the development of a novel in vitro method to induce human MDSC from healthy donor peripheral blood mononuclear cells (PBMC) by co-culture with human solid tumor cell lines. Suppressor cells generated by this method demonstrate features consistent with MDSC isolated from cancer patients, including the inhibition of autologous T cell responses to stimuli . Using this model system, we have determined the frequency of MDSC induction in human cancers of varied histiologic types, and have elucidated key tumor-derived factors that drive MDSC induction. Our methods generated highly purified human MDSC in quantities sufficient to enable robust morphology, phenotype, gene expression, and functional analyses. From these investigations two major subsets of MDSC have been identified that will help elucidate the role of these cells in the ontogeny, spread, and treatment of cancer.
Cell Lines and Cell Culture
Tumor cell lines were obtained from the American Type Culture Collection (ATCC) or were gifted to the Epstein laboratory. Tumor cell line authenticity was performed by cytogenetics and surface marker analysis performed at ATCC or in our laboratory. All cell lines were maintained at 37°C in complete medium [(RPMI-1640 with 10% fetal calf serum (characterized FCS, Hyclone, Inc., Logan, UT), 2 mM L-Glutamine, 100 U/mL Penicillin, and 100 μg/mL Streptomycin with 10 ng/mL hGM-CSF to support viability in co-cultures)], grown in tissue culture flasks in humidified, 5% CO2 incubators, and passaged 2-3 times per week by light trypsinization.
Tumor-Associated MDSC Generation Protocol
Human PBMC were isolated from healthy volunteer donors by venipuncture (60 mL total volume), followed by differential density gradient centrifugation (Ficoll Hypaque, Sigma, St. Louis, MO). PBMC were cultured in complete medium (5-10 × 105 cells/mL) in T-25 culture flasks with human tumor cell lines for one week. Tumor cells were seeded to achieve confluence by day 7 (approximately 1:100 ratio with PBMC), and samples in which tumor cells overgrew were excluded from analysis and were repeated with adjusted ratios. Alternatively, irradiated tumor cells (3500 rad) were initially seeded at a 1:10 ratio in co-cultures to examine whether induction was dependent upon actively dividing tumor cells. PBMC cultured in medium alone were run in parallel as an induction negative control for each donor to control for any effects of FCS. For these studies 39 male and 22 female healthy, volunteer donors ages 23 to 62 were used under USC Institutional Review Board-approved protocol HS-06-00579. Data were derived from at least two individuals and no inter-donor differences in MDSC induction or function were observed.
For antibody neutralization experiments, PBMC-tumor cell line co-cultures were repeated in the presence or absence of neutralizing monoclonal antibodies for a subset of HNSCC cell lines and included anti-VEGF (Avastin, Genetech, San Francisco, CA), anti-TNFα (Humira, Abbott, Abbott Park, IL), anti-IL-1β (clone AB-206-NA, Abcam, Cambridge, MA), anti-IL-6 (clone AB-201-NA, Abcam), anti-GM-CSF (clone BVD2), anti-TGFβ (clone 1D11), anti-FLT3L (polyclonal, Abcam), or isotype control. For cytokine induction, PBMC were cultured at 5-10 × 105 cells/mL in complete medium supplemented with 10 ng/mL GM-CSF, FLT3L (25 ng/mL, Abcam), and/or TGFβ (2 ng/mL, R&D).
ii. MDSC Isolation
After one week, all cells were collected from tumor-PBMC co-cultures. Adherent cells were removed using the non-protease cell detachment solution Detachin (GenLantis, San Diego, CA). Myeloid cells were then isolated from the co-cultures using anti-CD33 or anti-CD11b magnetic microbeads and LS column separation (Miltenyi Biotec, Germany) as per manufacturer's instructions. Purity of isolated cell populations was found to be greater than 90% by flow cytometry and morphological examination and viability of isolated cells was confirmed using trypan blue dye exclusion.
iii. Suppression Assay
The suppressive function of tumor-educated myeloid cells was measured by their ability to inhibit the proliferation of autologous T cells in the following Suppression Assay: T cells isolated from 30 mL of PBMC from returning healthy donors by anti-CD8 microbeads and magnetic column separation (Miltenyi Biotec) were CFSE-labeled (3 μM, Sigma) and seeded in 96-well plates with myeloid cells isolated previously (ii. MDSC isolation, above) at 2 × 105, cells/well 4:1 ratio. T cell proliferation was induce by anti-CD3/CD28 stimulation beads (Invitrogen, Carlsbad, CA). Suppression Assay wells were analyzed by flow cytometry for T cell proliferation after three days and supernatants were analyzed for IFNγ levels by ELISA (R&D Systems). Controls included a positive T cell proliferation control (T cells alone) and induction negative (medium only) and positive (GM-CSF + IL-6 cytokine-induced MDSC) controls . Where indicated specific inhibitors of MDSC were added to suppression assays including all-trans retinoic acid (ATRA, 100 nM, Sigma, St. Louis, MO), sunitinib (0.1 μg/mL, ChemieTek, Indiannapolis, IN), celecoxib (15 μM, Pfizer, New York, NY), nor- NOHA (500 μM, CalBiochem, San Diego, Ca), L-NMMA (500 μM, Calbiochem), apocynin (0.1 mM, Sigma), 1D11 antibody (10 μg/mL), SB431542 (5 μM, Tocris, Ellisville, MO), or Avastin (10 μg/mL, Genentech, San Francisco, CA). Samples were run in duplicate and data were collected as percent proliferation for 15,000 cells. Samples were run on a FACSCalibur flow cytometer (BD Biosciences, San Jose, CA) and data acquisition and analysis were performed using CellQuestPro software (BD) at the USC Flow Cytometry core facility.
Characterization of myeloid suppressor cells
i. Morphology of MDSC
Wright-Giemsa staining (Protocol Hema 3, Fisher, Kalamazoo, MI) of CD33+ or CD11b+ cell cytospin preparations was performed to assess the morphology of tumor-educated myeloid cells. Freshly isolated PBMC and CD33+ cultured in medium only or induced by cytokines GM-CSF + IL-6 were prepared in parallel for comparison. Observation, evaluation, and image acquisition were performed using a Leica DM2500 microscope (Leica Microsystems, http://www.leica-microsystems.com) connected to an automated, digital SPOT RTke camera and SPOT Advanced Software (SPOT Diagnostic Instrument Inc., http://www.diaginc.com). Images were resized for publication using Adobe Photoshop software (Adobe, http://www.adobe.com).
ii. Flow cytometry analyses of cell phenotypes
The phenotype of in vitro-generated MDSC was examined for expression of myeloid, antigen-presenting, and suppressor cell markers. For staining, cells were collected from flasks using Detachin to minimize cell surface protein digestion, and washed twice with FACS buffer (2% FCS in PBS) before resuspending 106 cells in 100 μl FACS buffer. Cells were stained for 1hr on ice with cocktails of fluorescently-conjugated monoclonal antibodies or isotype-matched controls, washed twice with FACS buffer, and resuspended in FACS buffer for analysis. For intracellular staining, cells were fixed and permeabilized using Fixation/Permeabilization Kit (eBioscience, San Diego, CA) after surface staining. Antibodies used were purchased either from BD Biosciences: CD11c (B-ly6), CD33 (HIM3-4), HLA-DR (L243), CD11b (ICRF44), CD66b (G10F5), CD14 (M5E2), CD68 (Y1/82A), 41BBL (C65-485), OX40L (Ik-1); or eBioscience: CD30 (Ber-H2), CD103 (B-Ly7), GITRL (eBioAITR-L), CD56 (MEM-188). Samples were run on a BD FACSCalibur flow cytometer and data acquisition and analysis were performed as above. Data are from three unique donors and expressed as a fraction of labeled cells within a live-cell gate set for 15,000 events. CD33+ or CD11b+ cells from PBMC cultured in medium alone were run in parallel for comparison.
iii. Real-time RT-PCR for gene expression of myeloid suppressor cells and tumor cell lines
For gene expression studies, tumor-educated CD33+ or CD11b+ cells were isolated from tumor-PBMC co-cultures by fluorescence activated cell sorting after Induction (i. Induction, above) and RNA was isolated from MDSC and DNase-treated using Qiagen's RNeasy micro kit. Tumor cells were collected from culture flasks and RNA isolated and DNase-treated using Qiagen's RNeasy mini kit. For real-time RT-PCR, 100ng of DNase-treated RNA was amplified with gene specific primers using one-step Power SYBR green RNA-to-Ct kit (Applied Biosystems) and run in an MX3000P Strategene thermocycler (La Jolla, CA). Data were acquired and analyzed using MxPro software (Stratagene). Gene expression was normalized to housekeeping gene GAPDH and fold change determined relative to expression levels in medium only-cultured cells. Primer sequences were obtained from the NIH qRT-PCR database http://primerdepot.nci.nih.gov and were synthesized by the USC Microchemical Core Facility .
iv. Measurement of tumor-derived factors by ELISA
Supernatants were collected from confluent cell line cultures, passed through a 0.2 μm syringe filter unit to remove cell debris, and stored in aliquots at -20°C. Levels of IL-1β, IL-6, TNFα, VEGF, and GM-CSF in supernatant samples were measured using ELISA DuoSet kits (R&D) per manufacturer's instructions. Plate absorbance was read on an ELX-800 plate reader (Bio-Tek, Winooski, VT) and analyzed using KC Junior software (Bio-Tek).
v. Functional studies
Tumor cell line-induced CD33+ or CD11b+ MDSC and medium only controls were isolated by magnetic bead separation (Miltenyi Biotec) and used for functional studies. Arginase activity was measured in cell lysates using Bioassay Systems' QuantiChrom Arginase Assay Kit (Hayward, CA) per the manufacturer instructions. For measurement of ROS production, freshly isolated myeloid cells were incubated for 45 minutes in RPMI with 3 μM DCFDA (Sigma) then analyzed by flow-cytometry. Nitrites were measured in supernatants of cells cultured 5 × 105 cells/mL overnight in complete medium using Promega's Griess Reagent System (Madison, WI) per the manufacturer instructions.
Immunohistochemistry studies were performed by the USC Department of Pathology Histology Core Facility (Los Angeles, CA) on cytospin preparations of suppressive and non-suppressive myeloid cells using antibodies against p-STAT3 (clone 6D779, dilution 1:400), C/EBPβ (clone H-7, dilution 1:100) (Santa Cruz Biotech), and HIF1a (clone 241812, dilution 1:50) (R&D Systems). Images were acquired and resized for publication as described above.
Changes in mean T cell proliferation and mean IFNγ production in the presence or absence of tumor-educated or cytokine-treated MDSC were tested for statistical significance by one-way ANOVAs followed by Dunnett test for pairwise comparisons of experimental samples to T cells alone. Changes in mean T cell proliferation in suppression assays in the presence or absence of single inhibitors of suppressive mechanisms were evaluated by ANOVA followed by Tukey's test for pairwise comparisons between all groups. Mean gene expression of 15 tumor-derived factors between HNSCC cell lines with and without CD33+ MDSC induction capacity was compared by ANOVA followed by Tukey's test for pairwise comparisons. For those factors with statistically significant different mean expression between suppressor cell inducing and non-inducing cell line groups, a linear regression analysis was performed to evaluate for a linear correlation between strength of suppressor cell induction and gene expression levels. Changes in mean T cell proliferation stimulated in the presence of suppressive CD33+ or CD11b+ cells induced by HNSCC or breast and lung carcinoma cell lines, respectively, for neutralization experiments were evaluated by ANOVA followed by Tukey's test for pairwise comparisons between all groups. Differences in mean expression of phenotypic markers between pooled groups of suppressive and non-suppressive CD33+ or CD11b+ cells were tested for significance by ANOVA followed by Bonferroni's multiple comparisons test for selected pairs (CD11b+ MDSC vs. CD11b+ medium control; CD33+ MDSC vs. CD33+ medium control). Differences in mean transcription factor or suppressive gene expression between CD11b+ and CD33+ MDSC were tested for significance by Student's t test. Differences in arginase activity, ROS production, and nitrite production among MDSC subsets and controls were evaluated by ANOVA followed by Bonferroni's multiple comparisons test for selected pairs (CD11b+ MDSC vs. CD33+ MDSC; CD11b+ MDSC vs. CD11b+ medium control; CD33+ MDSC vs. CD33+ medium control). Statistical tests were performed using GraphPad Prism software (La Jolla, CA) with a significance level of 0.05. Graphs and figures were produced using GraphPad Prism, Microsoft Excel, and Adobe Illustrator and Photoshop software (San Jose, CA).
Induction of tumor-associated human myeloid suppressor cells
Canonical CD33+ human MDSC induction by human cancer cell lines
Inducing Tumor Cell Line
Mean Percent Suppression
Inducing Tumor Cell Line
Mean Percent Suppression
T cells alone
** GM-CSF + IL-6
T cell Depl.
** USC-HN2 1
* SW 626
** SW 451
** SW 579
** DU 145
T cell Depl.
* SW 1353
** SW 1961
** SW 732
Non-small cell (2/7)
** SW 608
** SW 707
* SW 1573
** SW 1990
* Panc 2.03
* Panc 4.14
Small Cell 2/3)
* HA 22T
* HEP 3B
Strong CD33+ MDSC induction capability by a subset of human tumor cell lines
MDSC have been reported in patients with a wide range of different types of cancer [21–31] and their accumulation appears to correlate with increased tumor burden and stage [10, 30]. However, it remains unclear whether all cancers induce this tolerizing population, as strong evidence exists to suggest diversity in immune escape mechanisms amongst cancer types and individual tumors . To address this question, one-hundred-one human solid tumor cell lines were tested for their ability to induce MDSC in the tumor co-culture assay using PBMC from 61 unique healthy, volunteer donors (39 male, 22 female) ranging in age from 23-62 (Table 1). CD33+ MDSC could be generated by at least one cell line of every human tumor type examined (cervical/endometrial, ovarian, pancreatic, lung, head and neck, renal cell, liver, colorectal, prostate, thyroid, gastric, bladder, sarcoma, and glioblastoma), with the exception of breast carcinoma (Table 1). Head and neck, cervical/ovarian, colorectal, and renal cell carcinoma cell lines frequently induced CD33+ MDSC and are good models for further studies of this suppressive population. A range of suppressor cell ability appeared to exist within histologic types for the majority of tumor cell lines examined, suggesting that subclones within a whole tumor may drive MDSC induction. Notably, myeloid cells from PBMC cultured in medium alone or co-cultured with fibroblast cell lines were not suppressive (Table 1).
Tumor cell line-induced CD33+ MDSC resemble MDSC from cancer patients in suppressive function and gene expression
CD33+ MDSC are induced by tumor-derived IL-1β, IL-6, TNFα, VEGF, and GM-CSF
Preferential induction of a second subset of CD11b+ MDSC by some human cancer cell lines through FLT3L and TGFβ
Revisiting previously published gene expression data for this group of breast cancer cell lines, which lack CD33+ MDSC induction, we identified FLT3L and TGFβ as differentially expressed candidates for CD11b+ MDSC subset induction from our panel of putative MDSC-inducing factors . PBMC were then cultured in the presence of FLT3L, TGFβ, FLT3L + TGFβ, or medium alone for one week to evaluate whether these cytokines were sufficient for CD11b+ MDSC induction. Myeloid cells isolated from cytokine-treated cultures showed significant suppression of autologous T cell proliferation (p <0.05, comparison to T cells cultured alone), consistent with MDSC, with the most potent cells generated from combined FLT3L and TGFβ treatment (Figure 5B). These data suggest that FLT3L and TGFβ are present and sufficient for CD11b+ MDSC induction, but technical difficulties in abolishing FLT3L, which is a broad hematopoietic progenitor growth factor, and TGFβ, which is ubiquitous in serum and regulated by association of a latency protein, precluded clear neutralization data.
Characterization of human CD33+ and CD11b+ suppressor cells induced by tumor cell lines
Phenotype of MDSC shows CD33+ and CD11b+ subsets to be both HLA-DRlow and Lineage-
Comparison of suppressive function in CD33+ and CD11b+ MDSC subsets
A comparison of ARG-1, iNOS, and NOX2-component NCF1 gene expression in CD33+ and CD11b+ human MDSC induced by HNSCC or breast and lung carcinoma cell lines, respectively, revealed similar levels of expression between these subsets with a trend toward increased ARG-1 and NOX2 expression in CD33+ MDSC (Figure 6C). Functional studies confirmed greater arginase activity in CD33+ versus CD11b+ MDSC, but suggested that reactive oxygen species production is similarly elevated in both subsets (Figure 6D). Nitrite production was not found to be greatly elevated above medium only controls (data not shown), perhaps indicating that iNOS activity is a minor contributor for suppressive function in these subsets. While these findings remain preliminary, they suggest partial or complete functional overlap of these MDSC subsets. Furthermore, these data suggest that effective abrogation of human MDSC activities by depletion of a single subset is unlikely to yield significant therapeutic benefit in cancer patients that induce both subsets.
Higher Hif1α, STAT3, and C/EBPβ gene expression delineate subsets and distinguish tumor cell line-induced human MDSC from normal myeloid cells
It is apparent that human MDSC can be induced by multiple factors present in the tumor microenvironment . Furthermore, as a consequence of these multiple different induction routes, at least two distinct phenotypes of human MDSC emerge that can both mediate suppression of T cell responses. Interestingly, these CD33+ and CD11b+ MDSC subsets showed some phenotypic (HLA-DRlow and lineage-) and functional convergence despite preferential induction by different tumor models and predominant expression of either CD33 or CD11b. We wondered whether a common transcription factor was activated by these multiple pathways and might be act as a "master switch" to control both of these human MDSC. Several transcription factors have been proposed for control of MDSC, primarily in mice, including CCAAT-enhancer-binding proteins (C/EBP) β , hypoxia inducible factor (HIF) 1α , and signal transducer and activator of transcription (STAT) 3 [26, 38], STAT5 , and STAT6 . Previously identified as transcriptional regulators in some murine tumor-derived MDSC subsets, we now show that these transcription factors are elevated in human MDSC and, importantly, are differentially expressed in CD33+ versus CD11b+ MDSC subsets. We examined the expression of HIF1α, STAT3, and C/EBPβ in tumor cell line (SCCL-MT1 or USC-HN2)-induced CD33+ or (MCF7 breast or NCI-H60 small cell lung carcinoma) CD11b+ human suppressor cells compared with medium only controls by qRT-PCR techniques (data from six unique donors, two independent experiments) (Figure 8A) and immunohistochemistry (Figure 8B). Both CD33+ and CD11b+ functionally active human MDSC showed significant up-regulation of transcription factors STAT3, C/EBPβ, and HIF1α compared with non-suppressive myeloid cells from medium only cultures. However, CD33+ and CD11b+ MDSC subsets showed differences in transcriptional changes for these factors that were suggestive of different induction or activation pathways. As shown previously, CD33+ or CD11b+ MDSC may be induced under a variety of different tumor conditions and following incubation with several distinct cytokine mixtures . CD33+ MDSC showed stronger up-regulation of STAT3 and HIF1α while CD11b+ MDSC showed comparably greater up-regulation of C/EBPβ (Figure 8A). Differences in pSTAT3 and C/EBPβ were confirmed by immunohistochemistry studies (Figure 8B) and Western blotting techniques (data not shown) and preliminary data are shown for HIF1α protein accumulation to support gene expression findings. Treatment of either CD33+ or CD11b+ tumor-cell line-induced MDSC with lipopolysaccharide, a known activator of MDSC function , caused further up-regulation of STAT3, C/EBPβ, and HIF1α concurrent with increased expression of ARG-1, iNOS, and NOX2-component NCF1 (data not shown). These results further support a role for these transcription factors in promoting human MDSC suppressive function. While suppressive abilities in both CD11b+ and CD33+ subsets correlated with increased expression of STAT3, C/EBPβ, and HIF1α, the dominant transcriptional pathway may be different. Indeed, therapeutic reversal of CD11b+ or CD33+ MDSC-mediated suppression corresponded with different transcriptional changes.
Inhibitors of MDSC function show differential activity on MDSC subsets
As reviewed by Lechner and Epstein , tyrosine kinase inhibitor Sunitinib and all-trans retinoic acid (ATRA) have previously been shown to inhibit MDSC [26, 33]. Studies in our laboratory have also identified celecoxib (CXB) and analogs dimethyl celecoxib (DMC)  and unmethylated celecoxib (UMC)  as inhibitors of suppressive function in CD33+, but not CD11b+, MDSC in vitro (Figure 8C). Of note, the reversal of MDSC effects by CXB and analogs DMX and UMC does not appear to rely upon cyclo-oxygenase (COX)2 enzyme inactivation, as demonstrated by the persistence of therapeutic effects in the presence of prostaglandin E2 rescue, efficacy of analog DMC with low to absent COX inhibitory action, and the absence of effect seen with the structurally-unrelated COX2-selective inhibitor naproxen (Figure 8C). Gene expression patterns in ATRA, Sunitinb, or CXB-treated CD33+ or CD11b+ human MDSC were used to understand better factors promoting suppressive function in these cells. As shown in Figure 8D, functional inhibition of human CD33+ MDSC by ATRA, Sunitinib, and Celecoxib correlated with decreased STAT3 and HIF1α transcription. In comparison, functional inhibition of human CD11b+ MDSC by ATRA and Sunitinib correlated with decreased C/EBPβ levels, but no change in STAT3 and HIF1α mRNA levels. Celecoxib was not found to have inhibitory actions on CD11b+ MDSC and it was not observed to decrease C/EBPβ levels in this population. While preliminary, these data suggest that HIF1α, STAT3, and C/EBPβ may be key transcription factors related to suppressive function in tumor cell line-induced human MDSC, as was recently demonstrated for murine MDSC, and warrant further studies at the protein level as master regulators of suppressive activity with differential effects of human MDSC subsets.
Human MDSC comprise a diverse and complex group of suppressive cells that have been poorly characterized to date. Their accumulation and suppression of T cell responses in cancer patients, however, are quite clear and remain a barrier to successful cancer immunotherapy. In this study, using a new model for in vitro generation of tumor-associated human MDSC, we describe MDSC induction as a universal feature of human cancers and identify two distinct subsets of MDSC.
Studies to characterize human MDSC have been limited by the primary accumulation of these suppressor cells in individuals with significant illness (i.e. cancer, sepsis, trauma) and relative absence in healthy individuals . In our laboratory, induction of human MDSC from healthy donor PBMC by a one-week co-culture with select human cancer cell lines has allowed the generation of highly pure populations of MDSC in significant quantities for characterization studies and functional evaluation with autologous donor T cells. Using this induction method, we evaluated over 100 human solid tumor cell lines for the ability to induce canonical CD33+ human MDSC from healthy donor PBMC and found that these suppressor cells could be generated by tumor cell lines of all histiologic types, with the notable exception of breast carcinomas regardless of their HER2 and hormone receptor positivity. This finding prompted us to look for the induction of a different MDSC subset, and indeed we found that many tumor models with absent or poor CD33+ MDSC induction preferentially generated CD11b+ MDSC. Taken collectively, these data indicate that induction of MDSC is a common feature of human cancers and as such their presence may have a role in cancer detection and monitoring.
Using this model system, we then probed the pathways of induction and functional characteristics of these two cancer-associated MDSC subsets. Combining our previously published cytokine and gene expression data  with new gene expression, cytokine-induction, and antibody neutralization studies presented here, we identified IL-6, IL-1β and GM-CSF as the major inducing factors of CD33+ MDSC and FLT3L and TGFβ as major contributors to CD11b+ MDSC induction. Although generated by different tumor co-culture conditions, these two subsets appear to show at least partial overlap in morphology, phenotype, and function. Compared with their normal, non-suppressive myeloid counterparts, CD33+ and CD11b+ MDSC both showed immature myeloid morphology, low HLA-DR expression, and lacked lineage mature surface markers. MDSC have multiple mechanisms by which they can suppress T cell effector responses, and both CD33+ and CD11b+ subsets of MDSC showed up-regulation of canonical suppressive mechanisms (ARG-1, iNOS, NOX2). Previously, we demonstrated that subtle variations emerged in the patterns of suppressive genes that were up-regulated in human myeloid suppressor cells by different cytokine mixtures associated with active suppressive function . Similarly, human MDSC induced by a range of human solid tumor cell lines exhibited small differences in the up-regulation of suppressive genes that likely result from subsets within the broadly defined myeloid suppressor cell population. Of note, some tumor models were found to induce both CD33+ and CD11b+ MDSC subsets, while others induced only one or neither population. Stratification into CD11b+ and CD33+ subsets showed greater arginase activity in the CD33+ subset and partial overlap of function. These results likely reflect the complexity of myeloid suppressor cells, and will require finer dissection in future studies.
1Russell SM, Lechner MG, Gong L, Megiel C, Liebertz DJ, Masood R, Correa AJ, Han J, Puri JK, Sinha UK, Epstein AL. USC-HN2, a new model cell line for recurrent oral cavity squamous cell carcinoma, with immunosuppressive characteristics. Oral Oncology, in press.
- (C/EBP) β:
5- (and 6-): CCAAT/enhancer-binding protein
carboxyfluorescein diacetate succinimidyl ester
- (c-kit L):
c-kit ligand or stem cell factor
fms-related tyrosine kinase 3 ligand
glyceraldehyde 3-phosphate dehydrogenase
granulocyte-macrophage colony stimulating factor
hypoxia inducible factor-1 alpha
- indoleamine 2:
inducible nitric oxide synthase
macrophage colony stimulating factor
myeloid-derived suppressor cells
nuclear factor kappa B
peripheral blood mononuclear cells
regulatory T cells
Signal transducer and activator of transcription 3
transforming growth factor beta
tumor necrosis factor alpha
vascular endothelial growth factor-a.
Acknowledgements and Funding
The authors thank Dr. Dixon Gray for flow cytometry support, Lillian Young for performing immunohistochemistry studies, Dr. Daniel Liebertz for creation of the schematic in Figure 1, and Dr. Axel Schonthal for providing celecoxib analogs. Furthermore, the authors thank Dr. Adi Gadzar (UT Southwestern Medical Center, Dallas, TX), the Scott and White Clinic (Temple, TX) and Dr. Liz Jaffe (Johns Hopkins Medical Center, Baltimore, MD) for their generous contributions of cell lines used in these studies. This work was supported by National Institutes of Health Training Grant Award 3T32GM067587-07S1, USC Institute for Innovation Ideas Empowered Program, Philanthropic Educational Organization Scholars Award, and Cancer Therapeutics Laboratories, Inc. (Los Angeles, CA).
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