Myeloid-derived Suppressor Cells in Autoimmune Diabetes: Their Anti-diabetic Potential and Mechanism

Abbreviations: AID: Autoimmune Diabetes; MDSC: MyeloidDerived Suppressor Cells; NOD: Non-Obese Diabetic; B: Biobreeding LETL: Long Evans Tokushima Lean; APC: Antigen-Presenting Cells; Teff Cells: Effector T Cells; Treg Cells: Regulatory T Cells; MHC II: Histocompatibility Complex Class II; CLTA4: Cytotoxic T-Lymphocyte Antigen 4; IL: Interleukin; IFN: Interferon; TGF: Tumor Growth Factor; Inos: Inducible Nitric Oxide Synthase; NO: Nitric Oxide; TNF: Tumor Necrosis Factor; ROS: Reactive Oxygen Species; CCR2: CC Chemokine Receptor; CCL2: CC Chemokine Ligand; MMP: Matrix Metalloproteinase; TLR: Toll-Like Receptors; IMC: Immature Myeloid Cells; PIR: Paired Immunoglobulin-Like Receptors; ICAM-1: Intercellular Adhesion Molecule 1

The development of MDSC in different circumstances is not well understood [33]. In physiological conditions, Immature Myeloid Cells (IMC) differentiate from myeloid progenitors and, gradually, mature into dendritic cells, macrophages, and granulocytes/neutrophils upon migrating to the periphery (Figure 2A). In pathological conditions, abundant growth factors associated with diseases stimulate IMC expansion and subsequently, disturb their normal differentiation in bone marrow [34].
Mirroring the nomenclature of type 1 classic activation-like (M1) and type 2 alternative activation-like (M2) macrophages, polarized MDSC can be defined as M1 and M2 cells based on their corresponding phenotypes and functions ( Figure 2B). The molecular basis of MDSC development at the stages of expansion, activation, and functional polarization is largely unknown. One signal model was originally proposed to explain the requirement of one of tumor-associated factors for MDSC development.
More recently, this model was evolved into the "two signal model" stating that two distinct tumor-associated mediators are required at the stages of MDSC expansion and activation [33]. Since MDSC development from expansion to activation and functional polarization is a multiple-step process, "multiple signal model" in which multiple factors/signals are necessary for this process should be considered. However, type and mechanism of the pathology-associated factors in pathogenesis of MDSC from hematopoietic progenitors remain mostly unclear.
Functional polarization of MDSC is less studied probably due to the complexity and heterogeneity of MDSC subsets. Compelling evidence support the concept that tumor-associated MDSC predominantly exhibit M2-like phenotypes and immunosuppressive and pro-tumoral activities [28,31,32,34,36,[40][41][42]. However, co-existence of M1 and M2 phenotypes in MDSC was observed in few cases [43]. Genes, environment and immune dysregulation drive AID development. During disease development, antigen-presenting cells (APC) capture autoantigens, move to the pancreatic lymph nodes (PLN). After activation, Teff cells differentiate into different subsets, enter the pancreatic islets, release pro-inflammatory cytokines (IFN-γ, TNF-a and perforin) and destroy b-cells.
Eight intervention steps to prevent AID are proposed.
Step 2: Activation and differentiation of T cell subsets, respectively.
Step 3: Impairment of APC by MDSC.
Step 5: Induction of T reg by MDSC.
Step 6: T reg cell inhibition of Teff activation by IL-2 deprivation and B7 reduction.
Step 7: Reduction of TNF-a and IFN-γ production in Teff cells by T reg cells.
Step 8: Migration of immune cells into pancreatic islets. Arrow (thin line) and inhibitory sign (thick line) indicate promotion and suppression, respectively.
Polarization of MDSC involves an array of signaling cascades, leading to their acquisition of phenotypes and functionalities. Several studies showed that IFN-γ could induce iNOS expression whilst IL-4 or IL-13 increased arginase expression in MDSC as well as macrophages . Furthermore, activation from Toll-Like Receptors (TLR), IFN-γR, IL-4R, IL-13R could modify MDSC function [32,36,37,[50][51][52][53]. Consistently, membrane receptors such as TLR, Interferon-γReceptor (IFN-γR), Interleukin-4 Receptor (IL-4R) and IL-10R have been reported to participate in the function and expression of inducible Nitric Oxide Synthase (iNOS), Tumor Necrosis Factor (TNF)-α, M1 hallmarks, and arginase, M2 hallmarks [42,44]. Besides, studies in tumor-bearing hosts using pharmacological intervention and/or genetic ablation revealed that paired immunoglobulin-like receptors (PIR) and Tumor Growth Factor Β Receptor (TGF-βR) modulate the polarization of M-MDSC and G-MDSC, respectively (Figures 3A and 3B) [42,44]. Therefore, ligands, receptors, and downstream mediators of the PIR-B and TGF-βR pathways are potential targets for manipulation of functional phenotypes of MDSC that can be used for treatment of autoimmunity, cancer and other diseases. More information on the molecular basis of MDSC polarization and related functional changes is required for their further clinical applications.
Multiple mechanisms of MDSC in immune regulation: MDSC are one of the pivotal regulators of innate and adaptive immunity. They act as a "hub" to link and cross-talk with other immune cells in favor of immune tolerance in order to maintain disease progression and persistence. The details regarding the coordinated regulation of MDSC and other immune cells were summarized in Figure 4A [54,56,57]. MDSC can impair DC functions by decreasing maturation, antigen uptake and migration and skewing DC cytokine profile from inflammatory phenotype to anti-inflammatory one [58]. Additionally, MDSC interact with macrophages. MDSC diminish inflammation by downregulating macrophage production of IL-12, IL-6 and MHC II. This down-regulation appears to require IL-10 and cell contact [54]. MDSC also suppress development and function of NK cells and this suppression can be enhanced by inflammation [59,60]. As far as T cells are concerned, MDSC can induce T eff cell inactivation and apoptosis [61][62][63][64][65] and expand T reg cells [9,32,36,[66][67][68][69]. T cell suppression and T reg expansion by MDSC are cell contact-, NO-and/or arginasedependent [36,61,[70][71][72][73][74]. M2-like M-MDSC possess higher abilities to suppress T eff cell activation and proliferation than M1-like counterparts in the co-culture of T cells with M-MDSC and in vivo [42]. Moreover, M-MDSC with M2 functional phenotype possess higher potency in T reg expansion than those with M1 phenotype in vitro and in vivo [42]. M2 M-MDSC-induced T reg increase seemed to be IL-10, IL-4 and IL-13mediated arginase-dependent [42]. GMDSC could inhibit CD8 T cell activity in tumor-bearing hosts [44]. However, the ability of G-MDSC to induce T reg expansion is not corroborated. Overall, MDSC with M2 functional phenotype induce higher immune tolerance than those with M1 phenotype.

Potential and mode of action of MDSC in suppressing AID
MDSC have emerged as one of key immune regulators, raising a hypothesis that MDSC can treat AID and other autoimmune diseases. This hypothesis was first assessed in mouse models of AID as evidenced by two seminal studies [9,10]. One study from our group, for the first time, demonstrated that MDSC isolated from tumor-bearing mice mediated T reg induction or T eff suppression dependently on a MHC II-dependent antigen presentation [9]. The mechanism of action of MDSCs is via secretion of anti-inflammatory cytokines (TGF-β and IL-10), induction of CD4 + CD25 + Foxp3 + T reg or suppression of T eff proliferation that are beneficial for creating host immune tolerance [9]. To understand the role of MDSCs in murine diabetes models, we showed that adoptive transfer of MDSCs reduced diabetes by 75% compared with control group in RIP-HA/Rag2-/-mice [9]. Moreover, the protective role of MDSCs in NOD/SCID mice was investigated [9]. NOD/SCID mice were injected with diabetogenic T cells from diabetic NOD mice in the presence of MDSCs. Consistently, protective efficacy of MDSCs is dose-dependent and single dose treatment of MDSCs showed significant long-term protection, i.e. 60% remained diabetes free over 14-week observation [9]. The overall data prove the concept that MDSCs can suppress AID via regulation of T cell-mediated tolerance. It is worth mentioning that the MDSC were characterized as M2 MDSC. Later on, the other study confirmed the function of MDSC in AID development. They first showed that temporary B-cell depletion by anti-hCD20 antibody increased CD11b + Gr1 + splenocytes by 6% in h-CD20/NOD transgenic mice [10].
Next, they found that these myeloid cells inhibited T cell proliferation in vitro in a NO-and cell contact dependent fashion, suggesting that this subset had MDSC characteristics [10]. Strikingly, they were able to employ one single dose of anti-Gr1 antibody (RB6-8C5 clone) to induce a significant expansion of CD11b + Gr1 + cells in NOD mice whose diabetic incidence was reduced by ~40%. Besides, anti-TGF-β neutralizing antibody almost abolished the reduction of diabetic incidence in NOD mice, suggesting the implication of TGF-β in the function of CD11b + Gr1 + cells. The CD11b + Gr1 + cells showed perfect traits of MDSC as evidenced by in vitro T eff suppression and T reg induction assays [10]. Taken together, MDSC suppress AID via multiple mechanisms involving T eff inactivation, T reg induction, cell contact and soluble mediators (TGF-β, IL-10, NO, etc.) ( Figure 4B). Besides, MDSC polarization could affect a potency level of MDSC in AID prevention and/or therapy.
Several lines of evidence have proved the principle indicating a great potential of MDSC-based strategy for AID prevention in mouse models [9,10]. Clearly, immunotherapy with MDSC underscores the establishment of long-term immune tolerance before a complete destruction of remaining β-cells or β-cell replacement/regeneration in hosts, leading to the AID cure. However, such immunotherapy is a double-edged sword. On one hand, it can suppress aberrant autoimmunity. On the other hand, this therapy may increase the risk of infections and malignancy. Ideally, manipulating MDSC to establish antigen-specific immune tolerance can minimize the above risk, which was proven possible in the mouse model [9,10]. MDSCbased immunotherapy for AID from bench side to bed side needs to overcome several hurdles, i.e., reliable source of human MDSC, in vivo establishment of auto-antigen-specific immune tolerance by MDSC and re-establishment of MDSC induced immune tolerance after loss. Before fully exploiting MDSC for AID, more questions remain to be addressed, whether or not MDSC exert their action on macrophages, DC, B and NK cells in AID protection, the mechanism by which MDSC induce T reg cells, whether MDSC are effective for AID therapy, costeffective way of producing enough and safe MDSC for clinical trials, the relationship of MDSC polarization and AID prophylaxis/therapy and impact of MDSC on β-cell function.

Concluding Remarks
AID is an autoimmune endocrine disorder with premature death. Mounting data have clearly pointed to a critical role of MDSC in autoimmune diabetes. Although some advances have been made in  Figure 3A, signals from LPS/IFN-γ and IL-4/IL-13 can dictate G-MDSC polarization into G1 and G2 cells, respectively. Both cell types are characterized by G1 hallmarks (TNF-a, Fas, ICAM-1, and ROS) and G2 hallmarks (arginase, IL-10 and CCL2/5), respectively. TGF-b is known as a negative regulator of G-MDSC polarization (44)(45)(46). Upon TGF-b binding in most cell types, TGF-bRII/RI dimer forms and activates SMAD2/3, leading to the increase of SMAD7 expression and NF-κB inhibition (74). Current data support the concept that TGF-b inhibits G1 pathway but promotes G2 pathway. It is still unclear whether and how SAMD2/3 and SMAD7 mediated TGF-b-mediated G1/G2 polarization. Arrow (thin line) and inhibitory sign (thick line) indicate promotion and suppression, respectively.
understanding MDSC development from expansion, activation to polarization stages in recent years, relatively little is known about the multi-stage process. Here, we brought up an evolving concept of the multiple signal model in regulating MDSC development. Moreover, the signaling cascades involving PIR and TGF-β receptors were discussed for the polarization of M-MDSC and G-MDSC, respectively. Control over this polarization might have an impact on the clinical potential of MDSC in AID therapy. A special emphasis was placed on recent progress in understanding the therapeutic potential and mechanism of action of MDSC in AID. A new view on MDSC-based interference with AID development was also discussed.