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Suppression of T cells by myeloid-derived suppressor cells in cancer
Accepted Manuscript Suppression of T cells by myeloid-derived suppressor cells in cancer Jieying Chen, Yingnan Ye, Pengpeng Liu, Wenwen Yu, Feng Wei, Hui Li, Jinpu Yu PII: DOI: Reference:
S0198-8859(16)30515-8 http://dx.doi.org/10.1016/j.humimm.2016.12.001 HIM 9873
To appear in:
Human Immunology
Received Date: Revised Date: Accepted Date:
30 January 2016 2 December 2016 5 December 2016
Please cite this article as: Chen, J., Ye, Y., Liu, P., Yu, W., Wei, F., Li, H., Yu, J., Suppression of T cells by myeloidderived suppressor cells in cancer, Human Immunology (2016), doi: http://dx.doi.org/10.1016/j.humimm. 2016.12.001
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Suppression of T cells by myeloid-derived suppressor cells in cancer
Jieying Chen1, Yingnan Ye1, Pengpeng Liu2, Wenwen Yu1, Feng Wei1, Hui Li1, Jinpu Yu1, 2# 1
Department of Immunology, National Clinical Research Center of Cancer, Tianjin Key Laboratory of Cancer
Immunology and Biotherapy, Tianjin Medical University Cancer Institute and Hospital, Tianjin 300060, China; 2
Cancer Molecular Diagnostic Core, National Clinical Research Center of Caner, Tianjin Key Laboratory of
Cancer Prevention and Therapy, Tianjin Medical University Cancer Institute and Hospital, Tianjin 300060, China. #
Corresponding author: Tiyuanbei, Huanhuxi Road, Hexi District, Tianjin 300060, People’s Republic of China,
Email:[email protected]
Short title: Suppression of T cells by MDSCs in cancer
Competing interests: The authors declare that they have no conflict of interest.
1
Abstract: Myeloid-derived suppressor cells (MDSCs) are a population of immature myeloid cells defined by their immunosuppression. Elevated levels of certain soluble cytokines in tumor microenvironment, such as IL-6 and IL-10, contribute to the recruitment and accumulation of tumor-associated MDSCs. In turn, MDSCs secret IL-6 and IL-10 and form a positive self-feedback to promote self-expansion. MDSCs also release other soluble cytokines such as TGF-β and chemokines to exert their suppressive function by induction of regulatory T cells. Exhaustion of some amino acids by MDSCs with many secretory enzymes or membrane transporters as well as their metabolites leads to blockage of T cells development. The interaction of membrane molecules on MDSCs and T cells leads inactivation and apoptosis of T cells. There may be one or some dominant mechanism(s) by which MDSCs impair the immune system in different tumor microenvironment. Thus, it is important to identify the subpopulations of MDSCs and clarify the dominant mechanism(s) through which MDSCs inhibit antitumor immunity in order to establish a more individual immunotherapy by eliminating MDSCs-mediated suppression. Currently studies concentrated on therapeutic strategies targeting MDSCs have obtained promising results. However, more studies are needed to demonstrate their clinical safety and efficacy.
Key words: myeloid-derived suppressor cell; immune suppression; T cell; targeted therapy; cancer
2
Introduction Myeloid-derived suppressor cells (MDSCs) were first detected in mouse models bearing human tumors[1] before they were identified in patients with head and neck squamous cancer several years later[2]. Actually, MDSCs are a heterogeneous population of immature myeloid cells including granulocytes, macrophages and dendritic cells[3, 4], which display potent inhibitory effect on immunity and result in immune evasion. Commonly, murine MDSCs are characterized by the expression of Gr-1 and CD11b and represent approximately 2% to 4 % of all nucleated splenocytes, but can increase up to 50% in tumor bearing mice[5, 6]. The equivalent MDSCs in humans are usually positive for both CD11b and CD33 or express the CD33 but lack the expression of the major histocompatibility complex (MHC) class II molecule HLA-DR. They account for less than 0.5% of peripheral blood mononuclear cells in healthy individuals, but can increase more than 10 folds in circulation of cancer patients[7-10]. There are two main MDSC subtypes both in mice and humans, polymorphonuclear MDSC (PMN-MDSC) and monocytic MDSC (M-MDSC), in which PMN -MDSCs represent the major subset of circulating MDSC[11]. However, the specific markers for MDSC subsets, especially in humans, are vague until now and MDSCs are primarily identified by their suppressive function. Nowadays, it has been demonstrated that MDSCs are associated with poor prognosis in cancer patients[12], promote tumor angiogenesis[13], and inhibit both innate and adaptive immunity against tumors [14]. Depletion of MDSCs was reported to enhance the antigen presenting cell activity and increase the frequency and activity of the NK and T cell effectors in murine models of lung cancer[15]. Many studies have focused on the cancer-associated 3
immune-suppression mechanisms mediated by MDSCs[16, 17], in which inactivation of T cells is underscored. T cells represent a key effector arm of the immune system that is required for cancer control. Dysfunction of T cells fails to response to transformed cells, thus tumor-surveillance is impaired. Therefore, this review aims to introduce the underlying molecular mechanisms of MDSCs recruitment and their suppression on T cells, as well as current therapeutic strategies targeting MDSCs.
1
Soluble cytokines rich in tumor microenvironment recruit MDSCs and contribute to their suppressive function (Figure 1)
1.1 IL-6 IL-6 is a multifunctional cytokine which plays an important role in the regulation of the immune system. Although IL-6 was first considered as a potent pro-inflammatory cytokine, numerous studies have suggested that IL-6 plays a pivotal role in the pathological processes of numerous human cancers[18]. Increased levels of inflammatory cytokines including IL-6 have been reported in patients with various type of tumors[19], and an elevated level of IL-6 fostering progressive expansion of tumor cells[20] has been associated with poor clinical outcome[21-24]. Recently, more and more studies have demonstrated positive relationship between increased proportion of MDSCs and higher level of IL-6 in cancers [25, 24, 12]. By studying patients with gastrointestinal malignancies, Mundy-Bosse BL, et al [24] reported that plasma IL-6 level was correlated with CD33+HLA-DR-CD15+ MDSCs subsets, and the percentage of certain MDSC 4
subsets (CD15+ and CD15-) were inversely correlated with IFN-α-induced STAT1 phosphorylation in
CD4+ T cells. However, in Tsukamoto H’s study[25], they revealed that IL-6
derived from tumor-bearing mice recruited MDSC enabled MDSC attenuation of Th1 development but not suppression of primary T-cell activation. Effector CD4+ T cells sensitized by MDSC-derived IL-6 are defective in eliminating tumors because of their decreased ability to produce IFN-γand their dampened helper activity for cognate tumor-specific CD8+ T cells. A recent study[12] has reported that circulating CD11b+CD14+HLA-DR- cells were significantly increased and associated with serum IL-6 levels in esophageal squamous cell carcinoma patients, and IL-6 induced human functional MDSC in vitro displayed increased reactive oxygen species (ROS), arginase-1 (ARG-1) and p-STAT3, which are important to inhibit T cell effector function[26, 27]. These results indicate that increased IL-6 in tumor microenvironment facilitates the recruitment and suppressive function formation of tumor-associated MDSC.
1.2 IL-10 It was reported that increased MDSC level was correlated with higher level of circulating IL-10 in patients with anaplastic thyroid cancer[28]. In 2011, Hart KM et al[29] have reported that CD11b+ myeloid cells were the predominant producers of IL-10 in the ascites of ovarian tumor-bearing mice, and in turn IL-10 signaling through IL-10 receptor was a required component to establish the phenotypic and functional characteristics of MDSC in tumor-bearing hosts. Also, MDSC produced IL-10 could drive the development of a LAG-3 expressing population of T cells whose production of functional IFN-γ is impeded compared to the 5
LAG-3 negative cells. Thus the authors concluded that IL-10 could influence T cell phenotype and function through both IL-10-driven MDSC inhibition and direct IL-10 receptor ligation resulting in an ineffective T cell pool incapable of mounting an effective immune response against the tumor. More recently, a study[30] has demonstrated that MDSCs isolated from tumor-bearing histone deacetylase 11 (HDAC11)-knockout
mice were more suppressive and
released more IL-10 than that from wild-type tumor-bearing control. Yet earlier in 2009, this research group has proved that HDAC11 is the key negative regulator of IL-10 transcription in antigen presenting cells in human and mouse[31]. These suggest that the differentiation of myeloid cells in the tumor microenvironment is blocked by abnormal activation of HDAC11/IL10 pathway resulting in accumulation of suppressive MDSCs.
1.3 TGF-β β TGF-β family of proteins is a set of pleiotropic secreted signaling molecules with unique and potent immunoregulatory properties. Increased production and activation of latent TGF-β have been linked to immune defects associated with malignancies and autoimmune disorders[32]. In a study for characterization of MDSC subsets in patients with squamous cell carcinoma of the head and neck, the CD14+HLA-DR- MDSC subset was noted to be the highest in number and produced higher levels of TGF-β compared with other subsets[33]. The addition of anti-TGF-β monoclonal antibody in combination with other antibodies (anti-CD86 monoclonal antibody and anti-PD-L1 monoclonal antibody) partially restored T-cell proliferation and IFN-γ production. MDSCs can also induce other immunoregulatory cells with release of TGF-β to exert their 6
immunosuppressive function. Chen W et al[34] have reported that TGF-βinduced Foxp3 gene expression in T cell receptor-challenged CD4+CD25- peripheral naïve T cells, which were then transformed toward a regulatory T cell (Treg) phenotype with potent immunosuppressive potential. It was also reported that tumor cells recruited immature dendritic cells secreted bioactive TGF-βand then stimulated Tregs proliferation in murine model[35].
1.4. Chemokines In a recent study [36], tumor infiltrating M-MDSCs not only produced high levels of nitric oxide (NO) and ARG-1, but also greatly increased levels of the ligands of CCR5: CCL3, CCL4, and CCL5. Expression of CCR5 was preferentially detected on Tregs. In vitro, tumor-infiltrating M-MDSCs directly attracted high numbers of Tregs via CCR5, while intratumoral injection of CCL4 or CCL5 increased tumor-infiltrating Tregs, and deficiency of CCR5 led to their profound decrease.
2 T cells are impaired by MDSCs with exhaustion of certain amino acids and their metabolites (Figure 2)
2.1 L-arginine L-arginine is a conditionally essential amino acid for adult mammals used for the biosynthesis of protein, creatine and spermine. ARG-1 and induced nitric-oxide synthase (iNOS) are two different but related enzymes that are expressed highly in MDSCs and utilize L-arginine 7
to produce urea and NO, respectively[37]. In 2005, Zea AH and his colleagues [38] demonstrated, for the first time, the existence of suppressor myeloid cells producing ARG in cancer patients. They found that ARG activity was significantly increased in a specific subset of CD11b+CD14-CD15+ cells in the peripheral blood from patients with metastatic renal cell carcinoma accompanied with markedly decreased cytokine production and low levels of T cell receptor CD3ζ chain. Depletion of the CD11b+CD14- myeloid suppressor cells restored CD3ζ chain expression on T cell and stimulated cell proliferation. Indeed, expression of ARG-1 has been reported to down-regulate T cell receptor expression by decreasing CD3ζ-chain biosynthesis [39]. The diminished expression of CD3ζ protein is paralleled by a decrease in CD3ζ mRNA caused by a significantly shorter CD3ζ mRNA half-life [39]. This provokes an arrest of T cells in the G0–G1 phase of the cell cycle, associated with a deficiency of protein kinase complexes such as cyclin D3 and cyclin-dependent kinase 4 that are important for G1 phase progression[40, 41].
2.2 Tryptophan Tryptophan is an essential amino acid for T-cell proliferation. It was reported that in the tumor microenvironment at the presence of IFN-γ, MDSCs suppressed T cells infiltrated in tumors and tumor-draining lymph nodes through indoleamine 2,3-dioxygenase (IDO) hydrolyzing tryptophan, which is essential for T-cell proliferation and induces arrest T cells in G0 stage[42]. Kallberg E et al[43] have shown that IDO-producing MDSCs mediated immune suppression in the early stages of prostate cancer progression. Recently, a research group[44] has reported that STAT3-dependent IDO expression mediated immunosuppressive effects of MDSCs 8
in breast cancer, in which MDSCs dramatically inhibited the proliferation of T cells and Th1 cytokines secretion,
promoted the apoptosis of T cells and Th2 cytokines secretion.
Furthermore, they demonstrated that the STAT3-stimulated IDO upregulation was mediated by noncanonical NF-κB activation [45].
2.3 Phenylalanine Now it has been proved that IL4-inducible gene 1 (IL4I1) is a secreted L-phenylalanine oxidase expressed by myeloid-derived cells such as dendritic cells[46] or tumor-associated macrophages[47], which can produce hydrogen peroxide and phenylpyruvate by oxidative deamination of phenylalanine. It was reported that human IL4I1 was able to inhibit TCR ζ chain expression and T-lymphocytes proliferation via hydrogen peroxide production.
2.4 Cystine MDSCs also block T cell activation by sequestering cystine and limiting the availability of cysteine[48]. Cysteine is an essential amino acid for T cell activation because T cells lack cystathionase [49], which converts intracellular methionine to cysteine[50, 51], as well as an intact xc− transporter[52, 53], which imports disulfide-bonded cystine, and then reduced to cysteine[54, 55]. T cells depend on antigen presenting cells such as macrophages and dendritic cells to export cysteine which is imported by T cells via neutral amino acid transporter ASC [56-58]. MDSCs express xc− transporters to import cystine; but they do not express ASC transporters and thus no cysteine is exported. MDSCs can compete with antigen presenting cells 9
for extracellular cystine. Therefore, at the presence of MDSCs, antigen presenting cells released less cysteine and the extracellular pool of cysteine was reduced. Thus, MDSCs consume cystine and do not return cysteine to the microenvironment, thereby depriving T cells of cysteine and inhibiting their activation and function [48].
2.5 Metabolites of amino acids Oxidative metabolism of amino acids produces ROS, which might cause fatal damage to cells in case of excessive production. Study has shown that oxidative stress, caused by MDSCs, inhibited CD3 ζ-chain expression in T cells and antigen-induced cell proliferation [59]. Corzo et al[60] have identified that ROS was up-regulated in Gr-1+CD11b+ MDSCs isolated from seven different mice tumor models and CD11b+CD14-CD33+ MDSCs in patients with head and neck cancer. Production of ROS by MDSCs is mediated through NADPH oxidase 2 (NOX2), a membrane-bound enzyme complex which is assembled during a respiratory burst to catalyze the one-electron reduction of oxygen to superoxide anion using electrons provided by NADPH. With further investigation, Corzo et al[60] found that MDSCs aforementioned showed significantly higher expression of the NOX2 subunits p47phox and gp91phox compared to immature myeloid cells from tumor-free mice. In the absence of NOX2 activity, MDSCs lost the ability to control T-cell hyporesponsiveness and differentiated into mature dendritic cells. A recent study[61] also demonstrated that CD14+HLA-DR-/low cells, a MDSC subpopulation in non-small cell lung cancer patients, expressed the NOX component gp91phox and generated high level of ROS, and inactivation of ROS reversed MDSCs-mediated immunosuppressive capacity on T cells. 10
As mentioned above, MDSCs can produce NO via iNOS. NO alone could suppress T cells via a variety of different mechanisms, such as inhibition of the phosphorylation and activation of JAK3 and STAT5 transcription factors[62], as well as declination of MHC class II molecule expression[63] and induction of T-cell apoptosis[64]. Furthermore, the cooperation of ROS with NO forms peroxynitrite[65, 66], which leads to the nitration of tyrosines in the T-cell receptor–CD8 complex[67]. This reaction might affect the conformational flexibility of T-cell receptor-CD8 and its interaction with peptide-loaded MHC class I molecule, render cytotoxic CD8+ T cells unresponsive to antigen-specific stimulation[67]. Indeed, nitration inhibits the binding of processed peptides to tumor cell-associated MHC, and as a result, tumor cells become resistant to antigen-specific tumor infiltrated lymphocytes[68]. In addition, peroxynitrite leads to the nitration of CCL2 chemokines thereby inhibiting tumor infiltrated lymphocytes trafficking into the tumor, resulting in trapping of antigen-specific cytotoxic T cells in the tumor-surrounding stroma[69]. A recent study [70] has proved that PMN-MDSC impaired T cells through the production of peroxynitrites, while M-MDSC suppressed by the release of NO, which is in accordance with the conclusion of previous research [71-73].
3
Interaction of membrane molecules on MDSC and T cells mediates the anergy and apoptosis of T cells (Figure 3)
3.1 A disintegrin and metalloproteinase domain 17 ( ADAM17) L-selectin, also known as CD62L, expresses on and directs T cells homing to lymph nodes 11
and tumor sites, and this process is important for cell-mediated adaptive antitumor immunity and immune surveillance[74, 75]. Naive T cells are activated by antigens there and subsequently become effector T cells. ADAM17 is an enzyme that cleaves the ectodomain of L-selectin. MDSCs down-regulate L-selectin on naive T cells through their plasma membrane expression of ADAM17, thus inhibiting antitumor immunity[76].
3.2 Galectin-9 (Gal-9) T cell immunoglobulin and mucin domain-3 (Tim-3) is specifically expressed on IFN-γ-producing CD4+ Th1 and CD8+ T cytotoxic type 1 cells[77]. The interaction of Tim-3 with its ligand Gal-9 induces T cell death and terminates Th1 immunity [78]. Gal-9 was reported to be naturally expressed on mice CD11b+Ly6G+ cells[79]. Yet the interaction of Tim-3 and Gal-9 contributed to the expansion of mice CD11b+Ly6G+F4/80low MDSCs and thus regulated Th1 immunity.
3.3 Programmed death-ligand 1 (PD-L1) Besides, a recent report [80] has shown that the interaction of programmed cell death protein 1 (PD-1) on T cells and PD-L1 on MDSCs impaired the functional T cells in patients with relapsed cancer. Another study [81] has demonstrated that blockade of the contact between PD-1 on T cells and PD-L1 on MDSCs in the tumor microenvironment increased the proliferation and function of tumor antigen-specific effector CD8 (+) T cells, upregulated cytotoxic T cells signaling molecules, and generated T memory precursor cells. 12
4 Therapeutic strategies targeting MDSCs Because of their considerable contribution to the development, progression and metastasis of cancer, many investigators have focused on therapeutic strategies targeting MDSCs. In general, mechanisms of the experimental agents used in these research comprise eliminating MDSCs, reducing products of MDSCs and inducing MDSCs to differentiate into nonsuppressive mature myeloid cells. Yu Y et al reported that phosphatidylserine-targeting antibody could drive the differentiation of MDSCs into M1-like TAMs and functional dendritic cells using tumor-bearing mice[82]. Tasquinimod, a novel antitumor agent at an advanced stage of clinical trial for treatment of prostate cancer, was reported to induce less immunosuppressive MDSCs and more tumor-specific CD8(+) T cells in mouse tumor models[83]. Polyinosinic-polycytidylic acid, used to be a vaccine adjuvant, was poved to reduce the absolute number of MDSCs and modulate their immunosuppressive function in a murine model of breast cancer[84]. Other drugs recently demonstrated to be effective to decrease the frequency, diminish the suppressive effects of MDSCs, and/or improve anti-tumor function of T cells include all-trans retinoic acid[85], RA190[86], adenosine monophosphate-activated protein kinase activator[87], colony stimulating factor-1 receptor kinase inhibitor[88], α-Difluoromethylornithine[89], indomethacin[90], fatty acid oxidation inhibitor[91]. However, none of these drugs are tested to be safe and effective in clinical trials.
Conclusion 13
In conclusion, MDSCs are accumulated and activated in tumor microenviroment by series of cytokines. MDSCs induce dysfunction of T cells through direct or indirect mechanisms and prepare immunodeficient enviroment for the development and metastasis of cancer cells. Anyhow, MDSCs derived from different tumors display different phenotypes, hinting us that there may be one or some dominant mechanism(s) by which MDSCs impair the immune system in different tumor microenvironment. Indeed, it was reported that PMN-MDSCs suppressed T cells predominantly by production of peroxynitrite, while M-MDSCs suppressed T cells predominantly via expression of nitric oxide [70]. Therefore, it is important to identify the subpopulations of MDSCs and clarify the dominant mechanism(s) through which MDSCs inhibit antitumor immunity in order to
establish a more individual immunotherapy by eliminating
MDSCs-mediated suppression. However, research on therapeutic strategies targeting MDSCs is less reported relative to the work on suppression mechanisms. Though promising results were reported in these studies, it still needs a long way to achieve clinical application. Before that, work on comprehensive understanding of their immune inhibition mechanisms and development of more specific and effective targeted agents is still necessary. Acknowledgements This study was funded by Key Projects in the National Science & Technology Pillar Program (2013ZX09303001, 2015BAI12B12, and 2015BAI12B15), National Natural Science Foundation of China (81472473 and 81272360), Tianjin Municipal Commission of Science & Technology Key Research Program (13ZCZCSY20300). 14
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Cancer Immunol Res. 2015;3(11):1236-47.
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Figure legends Figure 1 Soluble cytokines rich in tumor microenvironment recruit MDSCs and contribute to their suppressive function. Elevated levels of IL-6 and IL-10 in tumor microenvironment contribute to the recruitment and accumulation of tumor-associated MDSCs. In turn, MDSCs secret IL-6 and IL-10 and form a positive self-feedback to promote self-expansion. MDSCs also release TGF-β and chemokines (CCLs) to exert their suppressive function by induction of regulatory T cells (Tregs).
Figure 2 T cells are impaired by MDSCs with exhaustion of certain amino acids and their metabolites. MDSCs secrete enzymes including arginase-1 (ARG-1), induced nitric-oxide synthase (iNOS), IL4-inducible gene 1 (IL4I1), and indoleamine 2, 3-dioxygenase (IDO) and consume L-arginine (L-arg), phenylalanine (Phe) and tryptophan (Try), which are essential for activation or proliferation of T cells.
MDSCs also block T cell activation by sequestering cystine with xc−
transporter, which imports disulfide-bonded cystine, and then reduced to cysteine. Normally, because T cells lack cystathionase, which converts intracellular methionine to cysteine, and intact xc− transporter, they can only acquire cysteine dependent on the neutral amino acid transporter ASC, which imports cysteine from surrounding environment. Antigen presenting cells (APCs) absorb extracellular cystine with xc− transporter and pump-out cysteine with ASC transporter. With the competence with MDSCs, APCs release less cysteine and the extracellular pool of cysteine is reduced. In addition, the metabolism of amino acids produces reactive oxygen species 21
(ROS) and reactive nitrogen species (RNS), which inhibit CD3ζ expression on T cells. Pheny: phenylpyruvate; N- formyl: n-formylkynurenine; NO: nitric oxide; ONOO-: peroxynitrite.
Figure 3 Interaction of molecules on MDSC and T cells mediates anergy and apoptosis of T cells. ADAM17: a disintegrin and metalloproteinase domain 17; Gal-9: galectin-9; PD-1: programmed cell death protein 1; PD-L1: programmed death-ligand 1; Tim-3: T cell immunoglobulin and mucin domain-3.
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S0198-8859(16)30515-8 http://dx.doi.org/10.1016/j.humimm.2016.12.001 HIM 9873
To appear in:
Human Immunology
Received Date: Revised Date: Accepted Date:
30 January 2016 2 December 2016 5 December 2016
Please cite this article as: Chen, J., Ye, Y., Liu, P., Yu, W., Wei, F., Li, H., Yu, J., Suppression of T cells by myeloidderived suppressor cells in cancer, Human Immunology (2016), doi: http://dx.doi.org/10.1016/j.humimm. 2016.12.001
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Suppression of T cells by myeloid-derived suppressor cells in cancer
Jieying Chen1, Yingnan Ye1, Pengpeng Liu2, Wenwen Yu1, Feng Wei1, Hui Li1, Jinpu Yu1, 2# 1
Department of Immunology, National Clinical Research Center of Cancer, Tianjin Key Laboratory of Cancer
Immunology and Biotherapy, Tianjin Medical University Cancer Institute and Hospital, Tianjin 300060, China; 2
Cancer Molecular Diagnostic Core, National Clinical Research Center of Caner, Tianjin Key Laboratory of
Cancer Prevention and Therapy, Tianjin Medical University Cancer Institute and Hospital, Tianjin 300060, China. #
Corresponding author: Tiyuanbei, Huanhuxi Road, Hexi District, Tianjin 300060, People’s Republic of China,
Email:[email protected]
Short title: Suppression of T cells by MDSCs in cancer
Competing interests: The authors declare that they have no conflict of interest.
1
Abstract: Myeloid-derived suppressor cells (MDSCs) are a population of immature myeloid cells defined by their immunosuppression. Elevated levels of certain soluble cytokines in tumor microenvironment, such as IL-6 and IL-10, contribute to the recruitment and accumulation of tumor-associated MDSCs. In turn, MDSCs secret IL-6 and IL-10 and form a positive self-feedback to promote self-expansion. MDSCs also release other soluble cytokines such as TGF-β and chemokines to exert their suppressive function by induction of regulatory T cells. Exhaustion of some amino acids by MDSCs with many secretory enzymes or membrane transporters as well as their metabolites leads to blockage of T cells development. The interaction of membrane molecules on MDSCs and T cells leads inactivation and apoptosis of T cells. There may be one or some dominant mechanism(s) by which MDSCs impair the immune system in different tumor microenvironment. Thus, it is important to identify the subpopulations of MDSCs and clarify the dominant mechanism(s) through which MDSCs inhibit antitumor immunity in order to establish a more individual immunotherapy by eliminating MDSCs-mediated suppression. Currently studies concentrated on therapeutic strategies targeting MDSCs have obtained promising results. However, more studies are needed to demonstrate their clinical safety and efficacy.
Key words: myeloid-derived suppressor cell; immune suppression; T cell; targeted therapy; cancer
2
Introduction Myeloid-derived suppressor cells (MDSCs) were first detected in mouse models bearing human tumors[1] before they were identified in patients with head and neck squamous cancer several years later[2]. Actually, MDSCs are a heterogeneous population of immature myeloid cells including granulocytes, macrophages and dendritic cells[3, 4], which display potent inhibitory effect on immunity and result in immune evasion. Commonly, murine MDSCs are characterized by the expression of Gr-1 and CD11b and represent approximately 2% to 4 % of all nucleated splenocytes, but can increase up to 50% in tumor bearing mice[5, 6]. The equivalent MDSCs in humans are usually positive for both CD11b and CD33 or express the CD33 but lack the expression of the major histocompatibility complex (MHC) class II molecule HLA-DR. They account for less than 0.5% of peripheral blood mononuclear cells in healthy individuals, but can increase more than 10 folds in circulation of cancer patients[7-10]. There are two main MDSC subtypes both in mice and humans, polymorphonuclear MDSC (PMN-MDSC) and monocytic MDSC (M-MDSC), in which PMN -MDSCs represent the major subset of circulating MDSC[11]. However, the specific markers for MDSC subsets, especially in humans, are vague until now and MDSCs are primarily identified by their suppressive function. Nowadays, it has been demonstrated that MDSCs are associated with poor prognosis in cancer patients[12], promote tumor angiogenesis[13], and inhibit both innate and adaptive immunity against tumors [14]. Depletion of MDSCs was reported to enhance the antigen presenting cell activity and increase the frequency and activity of the NK and T cell effectors in murine models of lung cancer[15]. Many studies have focused on the cancer-associated 3
immune-suppression mechanisms mediated by MDSCs[16, 17], in which inactivation of T cells is underscored. T cells represent a key effector arm of the immune system that is required for cancer control. Dysfunction of T cells fails to response to transformed cells, thus tumor-surveillance is impaired. Therefore, this review aims to introduce the underlying molecular mechanisms of MDSCs recruitment and their suppression on T cells, as well as current therapeutic strategies targeting MDSCs.
1
Soluble cytokines rich in tumor microenvironment recruit MDSCs and contribute to their suppressive function (Figure 1)
1.1 IL-6 IL-6 is a multifunctional cytokine which plays an important role in the regulation of the immune system. Although IL-6 was first considered as a potent pro-inflammatory cytokine, numerous studies have suggested that IL-6 plays a pivotal role in the pathological processes of numerous human cancers[18]. Increased levels of inflammatory cytokines including IL-6 have been reported in patients with various type of tumors[19], and an elevated level of IL-6 fostering progressive expansion of tumor cells[20] has been associated with poor clinical outcome[21-24]. Recently, more and more studies have demonstrated positive relationship between increased proportion of MDSCs and higher level of IL-6 in cancers [25, 24, 12]. By studying patients with gastrointestinal malignancies, Mundy-Bosse BL, et al [24] reported that plasma IL-6 level was correlated with CD33+HLA-DR-CD15+ MDSCs subsets, and the percentage of certain MDSC 4
subsets (CD15+ and CD15-) were inversely correlated with IFN-α-induced STAT1 phosphorylation in
CD4+ T cells. However, in Tsukamoto H’s study[25], they revealed that IL-6
derived from tumor-bearing mice recruited MDSC enabled MDSC attenuation of Th1 development but not suppression of primary T-cell activation. Effector CD4+ T cells sensitized by MDSC-derived IL-6 are defective in eliminating tumors because of their decreased ability to produce IFN-γand their dampened helper activity for cognate tumor-specific CD8+ T cells. A recent study[12] has reported that circulating CD11b+CD14+HLA-DR- cells were significantly increased and associated with serum IL-6 levels in esophageal squamous cell carcinoma patients, and IL-6 induced human functional MDSC in vitro displayed increased reactive oxygen species (ROS), arginase-1 (ARG-1) and p-STAT3, which are important to inhibit T cell effector function[26, 27]. These results indicate that increased IL-6 in tumor microenvironment facilitates the recruitment and suppressive function formation of tumor-associated MDSC.
1.2 IL-10 It was reported that increased MDSC level was correlated with higher level of circulating IL-10 in patients with anaplastic thyroid cancer[28]. In 2011, Hart KM et al[29] have reported that CD11b+ myeloid cells were the predominant producers of IL-10 in the ascites of ovarian tumor-bearing mice, and in turn IL-10 signaling through IL-10 receptor was a required component to establish the phenotypic and functional characteristics of MDSC in tumor-bearing hosts. Also, MDSC produced IL-10 could drive the development of a LAG-3 expressing population of T cells whose production of functional IFN-γ is impeded compared to the 5
LAG-3 negative cells. Thus the authors concluded that IL-10 could influence T cell phenotype and function through both IL-10-driven MDSC inhibition and direct IL-10 receptor ligation resulting in an ineffective T cell pool incapable of mounting an effective immune response against the tumor. More recently, a study[30] has demonstrated that MDSCs isolated from tumor-bearing histone deacetylase 11 (HDAC11)-knockout
mice were more suppressive and
released more IL-10 than that from wild-type tumor-bearing control. Yet earlier in 2009, this research group has proved that HDAC11 is the key negative regulator of IL-10 transcription in antigen presenting cells in human and mouse[31]. These suggest that the differentiation of myeloid cells in the tumor microenvironment is blocked by abnormal activation of HDAC11/IL10 pathway resulting in accumulation of suppressive MDSCs.
1.3 TGF-β β TGF-β family of proteins is a set of pleiotropic secreted signaling molecules with unique and potent immunoregulatory properties. Increased production and activation of latent TGF-β have been linked to immune defects associated with malignancies and autoimmune disorders[32]. In a study for characterization of MDSC subsets in patients with squamous cell carcinoma of the head and neck, the CD14+HLA-DR- MDSC subset was noted to be the highest in number and produced higher levels of TGF-β compared with other subsets[33]. The addition of anti-TGF-β monoclonal antibody in combination with other antibodies (anti-CD86 monoclonal antibody and anti-PD-L1 monoclonal antibody) partially restored T-cell proliferation and IFN-γ production. MDSCs can also induce other immunoregulatory cells with release of TGF-β to exert their 6
immunosuppressive function. Chen W et al[34] have reported that TGF-βinduced Foxp3 gene expression in T cell receptor-challenged CD4+CD25- peripheral naïve T cells, which were then transformed toward a regulatory T cell (Treg) phenotype with potent immunosuppressive potential. It was also reported that tumor cells recruited immature dendritic cells secreted bioactive TGF-βand then stimulated Tregs proliferation in murine model[35].
1.4. Chemokines In a recent study [36], tumor infiltrating M-MDSCs not only produced high levels of nitric oxide (NO) and ARG-1, but also greatly increased levels of the ligands of CCR5: CCL3, CCL4, and CCL5. Expression of CCR5 was preferentially detected on Tregs. In vitro, tumor-infiltrating M-MDSCs directly attracted high numbers of Tregs via CCR5, while intratumoral injection of CCL4 or CCL5 increased tumor-infiltrating Tregs, and deficiency of CCR5 led to their profound decrease.
2 T cells are impaired by MDSCs with exhaustion of certain amino acids and their metabolites (Figure 2)
2.1 L-arginine L-arginine is a conditionally essential amino acid for adult mammals used for the biosynthesis of protein, creatine and spermine. ARG-1 and induced nitric-oxide synthase (iNOS) are two different but related enzymes that are expressed highly in MDSCs and utilize L-arginine 7
to produce urea and NO, respectively[37]. In 2005, Zea AH and his colleagues [38] demonstrated, for the first time, the existence of suppressor myeloid cells producing ARG in cancer patients. They found that ARG activity was significantly increased in a specific subset of CD11b+CD14-CD15+ cells in the peripheral blood from patients with metastatic renal cell carcinoma accompanied with markedly decreased cytokine production and low levels of T cell receptor CD3ζ chain. Depletion of the CD11b+CD14- myeloid suppressor cells restored CD3ζ chain expression on T cell and stimulated cell proliferation. Indeed, expression of ARG-1 has been reported to down-regulate T cell receptor expression by decreasing CD3ζ-chain biosynthesis [39]. The diminished expression of CD3ζ protein is paralleled by a decrease in CD3ζ mRNA caused by a significantly shorter CD3ζ mRNA half-life [39]. This provokes an arrest of T cells in the G0–G1 phase of the cell cycle, associated with a deficiency of protein kinase complexes such as cyclin D3 and cyclin-dependent kinase 4 that are important for G1 phase progression[40, 41].
2.2 Tryptophan Tryptophan is an essential amino acid for T-cell proliferation. It was reported that in the tumor microenvironment at the presence of IFN-γ, MDSCs suppressed T cells infiltrated in tumors and tumor-draining lymph nodes through indoleamine 2,3-dioxygenase (IDO) hydrolyzing tryptophan, which is essential for T-cell proliferation and induces arrest T cells in G0 stage[42]. Kallberg E et al[43] have shown that IDO-producing MDSCs mediated immune suppression in the early stages of prostate cancer progression. Recently, a research group[44] has reported that STAT3-dependent IDO expression mediated immunosuppressive effects of MDSCs 8
in breast cancer, in which MDSCs dramatically inhibited the proliferation of T cells and Th1 cytokines secretion,
promoted the apoptosis of T cells and Th2 cytokines secretion.
Furthermore, they demonstrated that the STAT3-stimulated IDO upregulation was mediated by noncanonical NF-κB activation [45].
2.3 Phenylalanine Now it has been proved that IL4-inducible gene 1 (IL4I1) is a secreted L-phenylalanine oxidase expressed by myeloid-derived cells such as dendritic cells[46] or tumor-associated macrophages[47], which can produce hydrogen peroxide and phenylpyruvate by oxidative deamination of phenylalanine. It was reported that human IL4I1 was able to inhibit TCR ζ chain expression and T-lymphocytes proliferation via hydrogen peroxide production.
2.4 Cystine MDSCs also block T cell activation by sequestering cystine and limiting the availability of cysteine[48]. Cysteine is an essential amino acid for T cell activation because T cells lack cystathionase [49], which converts intracellular methionine to cysteine[50, 51], as well as an intact xc− transporter[52, 53], which imports disulfide-bonded cystine, and then reduced to cysteine[54, 55]. T cells depend on antigen presenting cells such as macrophages and dendritic cells to export cysteine which is imported by T cells via neutral amino acid transporter ASC [56-58]. MDSCs express xc− transporters to import cystine; but they do not express ASC transporters and thus no cysteine is exported. MDSCs can compete with antigen presenting cells 9
for extracellular cystine. Therefore, at the presence of MDSCs, antigen presenting cells released less cysteine and the extracellular pool of cysteine was reduced. Thus, MDSCs consume cystine and do not return cysteine to the microenvironment, thereby depriving T cells of cysteine and inhibiting their activation and function [48].
2.5 Metabolites of amino acids Oxidative metabolism of amino acids produces ROS, which might cause fatal damage to cells in case of excessive production. Study has shown that oxidative stress, caused by MDSCs, inhibited CD3 ζ-chain expression in T cells and antigen-induced cell proliferation [59]. Corzo et al[60] have identified that ROS was up-regulated in Gr-1+CD11b+ MDSCs isolated from seven different mice tumor models and CD11b+CD14-CD33+ MDSCs in patients with head and neck cancer. Production of ROS by MDSCs is mediated through NADPH oxidase 2 (NOX2), a membrane-bound enzyme complex which is assembled during a respiratory burst to catalyze the one-electron reduction of oxygen to superoxide anion using electrons provided by NADPH. With further investigation, Corzo et al[60] found that MDSCs aforementioned showed significantly higher expression of the NOX2 subunits p47phox and gp91phox compared to immature myeloid cells from tumor-free mice. In the absence of NOX2 activity, MDSCs lost the ability to control T-cell hyporesponsiveness and differentiated into mature dendritic cells. A recent study[61] also demonstrated that CD14+HLA-DR-/low cells, a MDSC subpopulation in non-small cell lung cancer patients, expressed the NOX component gp91phox and generated high level of ROS, and inactivation of ROS reversed MDSCs-mediated immunosuppressive capacity on T cells. 10
As mentioned above, MDSCs can produce NO via iNOS. NO alone could suppress T cells via a variety of different mechanisms, such as inhibition of the phosphorylation and activation of JAK3 and STAT5 transcription factors[62], as well as declination of MHC class II molecule expression[63] and induction of T-cell apoptosis[64]. Furthermore, the cooperation of ROS with NO forms peroxynitrite[65, 66], which leads to the nitration of tyrosines in the T-cell receptor–CD8 complex[67]. This reaction might affect the conformational flexibility of T-cell receptor-CD8 and its interaction with peptide-loaded MHC class I molecule, render cytotoxic CD8+ T cells unresponsive to antigen-specific stimulation[67]. Indeed, nitration inhibits the binding of processed peptides to tumor cell-associated MHC, and as a result, tumor cells become resistant to antigen-specific tumor infiltrated lymphocytes[68]. In addition, peroxynitrite leads to the nitration of CCL2 chemokines thereby inhibiting tumor infiltrated lymphocytes trafficking into the tumor, resulting in trapping of antigen-specific cytotoxic T cells in the tumor-surrounding stroma[69]. A recent study [70] has proved that PMN-MDSC impaired T cells through the production of peroxynitrites, while M-MDSC suppressed by the release of NO, which is in accordance with the conclusion of previous research [71-73].
3
Interaction of membrane molecules on MDSC and T cells mediates the anergy and apoptosis of T cells (Figure 3)
3.1 A disintegrin and metalloproteinase domain 17 ( ADAM17) L-selectin, also known as CD62L, expresses on and directs T cells homing to lymph nodes 11
and tumor sites, and this process is important for cell-mediated adaptive antitumor immunity and immune surveillance[74, 75]. Naive T cells are activated by antigens there and subsequently become effector T cells. ADAM17 is an enzyme that cleaves the ectodomain of L-selectin. MDSCs down-regulate L-selectin on naive T cells through their plasma membrane expression of ADAM17, thus inhibiting antitumor immunity[76].
3.2 Galectin-9 (Gal-9) T cell immunoglobulin and mucin domain-3 (Tim-3) is specifically expressed on IFN-γ-producing CD4+ Th1 and CD8+ T cytotoxic type 1 cells[77]. The interaction of Tim-3 with its ligand Gal-9 induces T cell death and terminates Th1 immunity [78]. Gal-9 was reported to be naturally expressed on mice CD11b+Ly6G+ cells[79]. Yet the interaction of Tim-3 and Gal-9 contributed to the expansion of mice CD11b+Ly6G+F4/80low MDSCs and thus regulated Th1 immunity.
3.3 Programmed death-ligand 1 (PD-L1) Besides, a recent report [80] has shown that the interaction of programmed cell death protein 1 (PD-1) on T cells and PD-L1 on MDSCs impaired the functional T cells in patients with relapsed cancer. Another study [81] has demonstrated that blockade of the contact between PD-1 on T cells and PD-L1 on MDSCs in the tumor microenvironment increased the proliferation and function of tumor antigen-specific effector CD8 (+) T cells, upregulated cytotoxic T cells signaling molecules, and generated T memory precursor cells. 12
4 Therapeutic strategies targeting MDSCs Because of their considerable contribution to the development, progression and metastasis of cancer, many investigators have focused on therapeutic strategies targeting MDSCs. In general, mechanisms of the experimental agents used in these research comprise eliminating MDSCs, reducing products of MDSCs and inducing MDSCs to differentiate into nonsuppressive mature myeloid cells. Yu Y et al reported that phosphatidylserine-targeting antibody could drive the differentiation of MDSCs into M1-like TAMs and functional dendritic cells using tumor-bearing mice[82]. Tasquinimod, a novel antitumor agent at an advanced stage of clinical trial for treatment of prostate cancer, was reported to induce less immunosuppressive MDSCs and more tumor-specific CD8(+) T cells in mouse tumor models[83]. Polyinosinic-polycytidylic acid, used to be a vaccine adjuvant, was poved to reduce the absolute number of MDSCs and modulate their immunosuppressive function in a murine model of breast cancer[84]. Other drugs recently demonstrated to be effective to decrease the frequency, diminish the suppressive effects of MDSCs, and/or improve anti-tumor function of T cells include all-trans retinoic acid[85], RA190[86], adenosine monophosphate-activated protein kinase activator[87], colony stimulating factor-1 receptor kinase inhibitor[88], α-Difluoromethylornithine[89], indomethacin[90], fatty acid oxidation inhibitor[91]. However, none of these drugs are tested to be safe and effective in clinical trials.
Conclusion 13
In conclusion, MDSCs are accumulated and activated in tumor microenviroment by series of cytokines. MDSCs induce dysfunction of T cells through direct or indirect mechanisms and prepare immunodeficient enviroment for the development and metastasis of cancer cells. Anyhow, MDSCs derived from different tumors display different phenotypes, hinting us that there may be one or some dominant mechanism(s) by which MDSCs impair the immune system in different tumor microenvironment. Indeed, it was reported that PMN-MDSCs suppressed T cells predominantly by production of peroxynitrite, while M-MDSCs suppressed T cells predominantly via expression of nitric oxide [70]. Therefore, it is important to identify the subpopulations of MDSCs and clarify the dominant mechanism(s) through which MDSCs inhibit antitumor immunity in order to
establish a more individual immunotherapy by eliminating
MDSCs-mediated suppression. However, research on therapeutic strategies targeting MDSCs is less reported relative to the work on suppression mechanisms. Though promising results were reported in these studies, it still needs a long way to achieve clinical application. Before that, work on comprehensive understanding of their immune inhibition mechanisms and development of more specific and effective targeted agents is still necessary. Acknowledgements This study was funded by Key Projects in the National Science & Technology Pillar Program (2013ZX09303001, 2015BAI12B12, and 2015BAI12B15), National Natural Science Foundation of China (81472473 and 81272360), Tianjin Municipal Commission of Science & Technology Key Research Program (13ZCZCSY20300). 14
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Figure legends Figure 1 Soluble cytokines rich in tumor microenvironment recruit MDSCs and contribute to their suppressive function. Elevated levels of IL-6 and IL-10 in tumor microenvironment contribute to the recruitment and accumulation of tumor-associated MDSCs. In turn, MDSCs secret IL-6 and IL-10 and form a positive self-feedback to promote self-expansion. MDSCs also release TGF-β and chemokines (CCLs) to exert their suppressive function by induction of regulatory T cells (Tregs).
Figure 2 T cells are impaired by MDSCs with exhaustion of certain amino acids and their metabolites. MDSCs secrete enzymes including arginase-1 (ARG-1), induced nitric-oxide synthase (iNOS), IL4-inducible gene 1 (IL4I1), and indoleamine 2, 3-dioxygenase (IDO) and consume L-arginine (L-arg), phenylalanine (Phe) and tryptophan (Try), which are essential for activation or proliferation of T cells.
MDSCs also block T cell activation by sequestering cystine with xc−
transporter, which imports disulfide-bonded cystine, and then reduced to cysteine. Normally, because T cells lack cystathionase, which converts intracellular methionine to cysteine, and intact xc− transporter, they can only acquire cysteine dependent on the neutral amino acid transporter ASC, which imports cysteine from surrounding environment. Antigen presenting cells (APCs) absorb extracellular cystine with xc− transporter and pump-out cysteine with ASC transporter. With the competence with MDSCs, APCs release less cysteine and the extracellular pool of cysteine is reduced. In addition, the metabolism of amino acids produces reactive oxygen species 21
(ROS) and reactive nitrogen species (RNS), which inhibit CD3ζ expression on T cells. Pheny: phenylpyruvate; N- formyl: n-formylkynurenine; NO: nitric oxide; ONOO-: peroxynitrite.
Figure 3 Interaction of molecules on MDSC and T cells mediates anergy and apoptosis of T cells. ADAM17: a disintegrin and metalloproteinase domain 17; Gal-9: galectin-9; PD-1: programmed cell death protein 1; PD-L1: programmed death-ligand 1; Tim-3: T cell immunoglobulin and mucin domain-3.
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