Adoptive Transfer Of Myeloid-derived Suppressor Cells Research Articles

Inducible nitric oxide synthase-expressing myeloid-derived suppressor cells regulated by interleukin 35 contribute to the pathogenesis of psoriasis.

Although psoriasis is classified as a T cell-mediated inflammatory disease, the contribution of myeloid cells to the pathogenesis of psoriasis is not fully understood. In the present study, we demonstrated that the expression of the anti-inflammatory cytokine interleukin-35 (IL-35) was significantly increased in patients with psoriasis with a marked increase in the number of myeloid-derived suppressor cells (MDSCs). Similar results were obtained in an imiquimod-induced psoriasis mouse model. IL-35 reduced the total number of MDSCs and their subtypes in the spleens and psoriatic skin lesions, ameliorating psoriasis. IL-35 also reduced the expression of inducible nitric oxide synthase in MDSCs, although it had no significant effect on interleukin-10 expression. Adoptive transfer of MDSCs from imiquimod-challenged mice aggravated the disease and weakened the effect of IL-35 in the recipient mice. In addition, mice transferred with MDSCs isolated from inducible nitric oxide synthase knockout mice had milder disease than those with wild-type MDSCs. Furthermore, wild-type MDSCs reversed the effects of IL-35, while MDSCs isolated from inducible nitric oxide synthase knockout mice did not affect IL-35 treatment. In summary, IL-35 may play a critical role in the regulation of iNOS-expressing MDSCs in the pathogenesis of psoriasis, highlighting IL-35 as a novel therapeutic strategy for patients with chronic psoriasis or other cutaneous inflammatory diseases.

Calcium/Calmodulin Dependent Protein Kinase Kinase 2 Regulates the Expansion of Tumor-Induced Myeloid-Derived Suppressor Cells.

Myeloid-derived suppressor cells (MDSCs) are a hetero geneous group of cells, which can suppress the immune response, promote tumor progression and impair the efficacy of immunotherapies. Consequently, the pharmacological targeting of MDSC is emerging as a new immunotherapeutic strategy to stimulate the natural anti-tumor immune response and potentiate the efficacy of immunotherapies. Herein, we leveraged genetically modified models and a small molecule inhibitor to validate Calcium-Calmodulin Kinase Kinase 2 (CaMKK2) as a druggable target to control MDSC accumulation in tumor-bearing mice. The results indicated that deletion of CaMKK2 in the host attenuated the growth of engrafted tumor cells, and this phenomenon was associated with increased antitumor T cell response and decreased accumulation of MDSC. The adoptive transfer of MDSC was sufficient to restore the ability of the tumor to grow in Camkk2-/- mice, confirming the key role of MDSC in the mechanism of tumor rejection. In vitro studies indicated that blocking of CaMKK2 is sufficient to impair the yield of MDSC. Surprisingly, MDSC generated from Camkk2-/- bone marrow cells also showed a higher ability to terminally differentiate toward more immunogenic cell types (e.g inflammatory macrophages and dendritic cells) compared to wild type (WT). Higher intracellular levels of reactive oxygen species (ROS) accumulated in Camkk2-/- MDSC, increasing their susceptibility to apoptosis and promoting their terminal differentiation toward more mature myeloid cells. Mechanistic studies indicated that AMP-activated protein kinase (AMPK), which is a known CaMKK2 proximal target controlling the oxidative stress response, fine-tunes ROS accumulation in MDSC. Accordingly, failure to activate the CaMKK2-AMPK axis can account for the elevated ROS levels in Camkk2-/- MDSC. These results highlight CaMKK2 as an important regulator of the MDSC lifecycle, identifying this kinase as a new druggable target to restrain MDSC expansion and enhance the efficacy of anti-tumor immunotherapy.

Chronic stress promotes breast carcinoma metastasis by accumulating myeloid-derived suppressor cells through activating β-adrenergic signaling

ABSTRACT Numerous studies have found that chronic stress could promote tumor progression and this may be related to inhibtion of immune system. Myeloid-derived suppressor cells (MDSCs) are a heterogeneous population of cells with immunosuppressive activity. MDSCs may represent a key link between chronic stress and tumor progression. However, the role of stress-induced MDSCs in breast cancer progression is unclear. The present study showed that pre-exposure of chronic stress could lead to MDSCs elevation and facilitated breast cancer metastasis in tumor-bearing mice. Adoptive transfer of MDSCs could significantly increase lung metastatic foci. In contrast, lung metastasis could be alleviated by depleting endogenous MDSCs with Gr-1 antibody. The concentration of norepinephrine in serum and the expression of tyrosine hydroxylase in bone marrow could be significantly elevated by chronic stress. Moreover, propranolol, an inhibitor of β-adrenergic signaling, could inhibit breast carcinoma metastasis and prevent the expansion of chronic stress-induced MDSCs. Further study revealed that the expressions of IL-6 and JAK/STAT3 signaling pathways were upregulated by chronic stress in mice, and this upregulation could be inhibited by propranolol. Blocking the IL-6 signal or inhibiting the activation of the JAK/STAT3 signaling pathway could reduce tumor growth and metastasis by attenuating the accumulation of MDSCs in vivo. Besides, propranolol inhibited the expression of IL-6 in supernatant of 4T1 cells induced by isoproterenol and reduced the proportion of inducible MDSCs in vitro. Taken together, these data indicated that chronic stress may accumulate MDSCs via activation of β-adrenergic signaling and IL-6/STAT3 pathway, thereby promoting breast carcinoma metastasis.

Myeloid-Derived Suppressor Cells: Ductile Targets in Disease.

Myeloid-derived suppressor cells (MDSCs) represent a heterogeneous population of immature myeloid cells with major regulatory functions and rise during pathological conditions, including cancer, infections and autoimmune conditions. MDSC expansion is generally linked to inflammatory processes that emerge in response to stable immunological stress, which alter both magnitude and quality of the myelopoietic output. Inability to reinstate physiological myelopoiesis would fall in an “emergency state” that perpetually reprograms myeloid cells toward suppressive functions. While differentiation and reprogramming of myeloid cells toward an immunosuppressive phenotype can be considered the result of a multistep process that originates in the bone marrow and culminates in the tumor microenvironment, the identification of its driving events may offer potential therapeutic approaches in different pathologies. Indeed, whereas expansion of MDSCs, in both murine and human tumor bearers, results in reduced immune surveillance and antitumor cytotoxicity, placing an obstacle to the effectiveness of anticancer therapies, adoptive transfer of MDSCs has shown therapeutic benefits in autoimmune disorders. Here, we describe relevant mechanisms of myeloid cell reprogramming leading to generation of suppressive MDSCs and discuss their therapeutic ductility in disease.

MDSCs drive the process of endometriosis by enhancing angiogenesis and are a new potential therapeutic target.

Endometriosis affects women of reproductive age via unclear immunological mechanism(s). Myeloid-derived suppressor cells (MDSCs) are a heterogeneous group of myeloid cells with potent immunosuppressive and angiogenic properties. Here, we found MDSCs significantly increased in the peripheral blood of patients with endometriosis and in the peritoneal cavity of a mouse model of surgically induced endometriosis. Majority of MDSCs were granulocytic, produced ROS, and arginase, and suppressed T-cell proliferation. Depletion of MDSCs by antiGr-1 antibody dramatically suppressed development of endometrial lesions in mice. The chemokines CXCL1, 2, and 5 were expressed at sites of lesion while MDSCs expressed CXCR-2. These CXC-chemokines promoted MDSC migration toward endometriotic implants both in vitro and in vivo. Also, CXCR2-deficient mice show significantly decreased MDSC induction, endometrial lesions, and angiogenesis. Importantly, adoptive transfer of MDSCs into CXCR2-KO mice restored endometriotic growth and angiogenesis. Together, this study demonstrates that MDSCs play a role in the pathogenesis of endometriosis and identifies a novel CXC-chemokine and receptor for the recruitment of MDSCs, thereby providing a potential target for endometriosis treatment.

Myeloid-Derived Suppressor Cells Ameliorate Cyclosporine A-Induced Hypertension in Mice.

The calcineurin inhibitor cyclosporine A (CsA) suppresses the immune system but promotes hypertension, vascular dysfunction, and renal damage. CsA decreases regulatory T cells and this contributes to the development of hypertension. However, CsA's effects on another important regulatory immune cell subset, myeloid-derived suppressor cells (MDSCs), is unknown. We hypothesized that augmenting MDSCs would ameliorate the CsA-induced hypertension and vascular and renal injury and dysfunction and that CsA reduces MDSCs in mice. Daily interleukin-33 treatment, which increased MDSC levels, completely prevented CsA-induced hypertension and vascular and renal toxicity. Adoptive transfer of MDSCs from control mice into CsA-treated mice after hypertension was established dose-dependently reduced blood pressure and vascular and glomerular injury. CsA treatment of aortas and kidneys isolated from control mice for 24 hours decreased relaxation responses and increased inflammation, respectively, and these effects were prevented by the presence of MDSCs. MDSCs also prevented the CsA-induced increase in fibronectin in microvascular and glomerular endothelial cells. Last, CsA dose-dependently reduced the number of MDSCs by inhibiting calcineurin and preventing cell proliferation, as other direct calcineurin signaling pathway inhibitors had the same dose-dependent effect. These data suggest that augmenting MDSCs can reduce the cardiovascular and renal toxicity and hypertension caused by CsA.

Bidirectional immune tolerance in nonmyeloablative MHC-mismatched BMT for murine β-thalassemia

AbstractNonmyeloablative conditioning using total lymphoid irradiation (TLI) and rabbit antithymocyte serum (ATS) (the murine preclinical equivalent of antithymocyte globulin [ATG]) facilitates immune tolerance after bone marrow transplantation (BMT) across major histocompatibility complex (MHC) disparities and may be a useful strategy for nonmalignant disorders. We previously reported that donor effector T-cell function and graft-versus-host disease (GVHD) are regulated via recipient invariant natural killer T-cell (iNKT) interleukin-4–driven expansion of donor Foxp3+ naturally occurring regulatory T cells (Tregs). This occurs via recipient iNKT- and STAT6-dependent expansion of recipient myeloid dendritic cells (MDCs) that induce contact-dependent expansion of donor Treg through PD-1/PD ligand signaling. After TLI/ATS + BMT, Gr-1lowCD11c+ MDCs and Gr-1highCD11cneg myeloid-derived suppressor cells (MDSCs) were enriched in GVHD target organs. We now report that the recovery of both recipient MDSCs (P < .01) and MDCs (P < .01) is significantly increased when the alkylator cyclophosphamide (CTX) is added to TLI/ATS conditioning. In a BALB/c → B6 lethal GVHD model, adoptive transfer of MDSCs from TLI/ATS/CTX-conditioned recipients is associated with significantly improved GVHD colitis and survival (P < .001), conversion of MDSCs to PD ligand–expressing MDCs, and increased donor naturally occurring Treg recovery (P < .01) compared with control treatment. Using BALB/c donors and β-thalassemic HW-80 recipients, we found significantly improved rates of engraftment and GVHD following TLI/ATS/CTX compared with TLI/ATS, lethal or sublethal total body irradiation/ATS/CTX, or CTX/ATS conditioning. These data provide preclinical support for trials of TLI/ATG/alkylator regimens for MHC-mismatched BMT for hemoglobinopathies. The data also delineate innate immune mechanisms by which TLI/ATS/CTX conditioning may augment transplantation tolerance.

Myeloid-Derived Suppressor Cells Are Controlled by Regulatory T Cells via TGF-β during Murine Colitis

Myeloid-derived suppressor cells (MDSCs) are well known regulators of regulatory Tcells (Treg cells); however, the direct regulation of MDSCs by Treg cells has not been well characterized. We find thatcolitis caused by functional deficiency of Treg cells leads to altered expansion and reduced function ofMDSCs. During differentiation of MDSCs invitro from bone marrow cells, Treg cells enhanced MDSC function and controlled their differentiation through a mechanism involving transforming growth factor-β (TGF-β). TGF-β-deficient Treg cells were not able to regulate MDSC function in an experimentally induced model of colitis. Finally, we evaluated the therapeutic effect of TGF-β-mediated in-vitro-differentiated MDSCs on colitis. Adoptive transfer of MDSCs that differentiated with TGF-β led to better colitis prevention than the transfer of MDSCs that differentiated without TGF-β. Our results demonstrate an interaction between Treg cells and MDSCs that contributes to the regulation of MDSC proliferation and the acquisition of immunosuppressive functions.

CD11b+Gr-1+ myeloid-derived suppressor cells reduce atherosclerotic lesion development in LDLr deficient mice.

Myeloid-derived suppressor cells (MDSCs) form a heterogeneous population of cells composed of early myeloid progenitor cells and immature myeloid cells, which strongly suppress pro-inflammatory immune cells in inflammatory diseases. Currently, it is unknown whether MDSCs contribute to atherosclerosis, a chronic inflammatory disease in which accumulation of lipoproteins in the arterial wall activates the immune system causing abnormal vascular remodelling and vessel occlusion. Here, we investigated whether and how MDSCs contribute to the development of atherosclerosis. We show that MDSCs arise in the bone marrow of LDLr(-/-) mice during atherosclerosis and strongly suppress proliferation of T cells. Adoptive transfer of MDSCs into both female and male LDLr(-/-) mice fed a Western-type diet (WTD) ameliorates atherosclerosis with 35%. We observed a 54% reduction in adventitial T cells, and more specifically, MDSCs suppress Th1 and Th17 cells. In addition, treatment with MDSCs reduces circulating pro-atherogenic B2 cells. We found two subsets of MDSCs in the bone marrow of hypercholesterolemic mice, monocytic and granulocytic MDSCs (mo- and gr-MDSCs, respectively), of which the percentage of mo-MDSCs significantly increased during WTD feeding. Moreover, mo-MDSCs completely abolished splenocyte proliferation, whereas gr-MDSCs were unable to suppress proliferation. Mechanistically, we show that MDSCs from atherosclerotic mice suppress T cells in an IFN-γ- and nitric oxide-dependent manner, which is associated with the action of mo-MDSCs. This study demonstrates that MDSCs develop during atherosclerosis and reduce atherosclerosis via suppression of pro-inflammatory immune responses.

Abstract P183: Myeloid-Derived Suppressor Cells Prevent Cyclosporine A-Induced Hypertension, Endothelial Dysfunction, and Renal Injury in Mice

Cyclosporine A (CsA) is an immunosuppressive drug used to treat focal segmental glomerulosclerosis, reduce autoimmune diseases, and prevent allograft rejection; however a limitation of CsA is that it can induce vascular and renal injury as well as hypertension. Myeloid-derived suppressor cells (MDSCs) are a heterogeneous population of immature granulocytes, macrophages, and dendritic cells that suppress pro-inflammatory immune responses by secreting anti-inflammatory cytokines, inhibiting innate and adaptive immune cells, and inducing regulatory T cells (Tregs). We hypothesized that adoptive transfer of MDSCs would prevent CsA-induced vascular and renal injury and hypertension. Daily treatment of male C57BL6/J mice for 1 week with CsA (50 mg/kg/day, i.p. injection) significantly increased systolic blood pressure (Day 7 SBP in mmHg: Con=97±2 vs. CsA=145±3, p&lt;0.05 vs. Con), decreased aortic endothelium-dependent relaxation responses, increased aortic fibronectin levels, increased renal glomerular mesangial expansion, increased renal fibronectin levels, and decreased splenic Treg levels. Adoptive transfer of 1 million MDSCs by i.p. injection on days 1, 4, and 7 partially prevented the CsA-induced rise in systolic blood pressure (CsA+1M MDSCs=120±3 mmHg) and the detrimental vascular and renal effects. However, adoptive transfer of 2 million MDSCs fully prevented the CsA-induced hypertension (CsA+2M MDSCs=105±1 mmHg) and vascular and renal effects. The anti-hypertensive and vascular and renal protective effects of 2 million MDSCs were independent of Treg induction as splenic Treg levels remained significantly decreased. These data suggest that MDSCs can prevent the hypertension and toxicity caused by CsA independent of Tregs and may be a therapeutic target for CsA-treated patients.

Splenectomy inhibits non-small cell lung cancer growth by modulating anti-tumor adaptive and innate immune response

It has been shown that inhibitors of the immune system reside in the spleen and inhibit the endogenous antitumor effects of the immune system. We hypothesized that splenectomy would inhibit the growth of relatively large non-small lung cancer (NSCLC) tumors by modulating the systemic inhibition of the immune system, and in particular Myeloid Derived Suppressor Cells (MDSC). The effect of splenectomy was evaluated in several murine lung cancer models. We found that splenectomy reduces tumor growth and the development of lung metastases, but only in advanced tumors. In immune-deficient NOD-SCID mice the effect of splenectomy on tumor growth and metastatic spread disappeared. Splenectomy significantly reduced the presence of MDSC, and especially monocytic-MDSC in the circulation and inside the tumor. Specific reduction of the CCR2+ subset of monocytic MDSC was demonstrated, and the importance of the CCL2-CCR2 axis was further shown by a marked reduction in CCL2 following splenectomy. These changes were followed by changes in the macrophages contents of the tumors to become more antitumorigenic, and by increased activation of CD8+ Cytotoxic T-cells (CTL). By MDSC depletion, and adoptive transfer of MDSCs, we demonstrated that the effect of splenectomy on tumor growth was substantially mediated by MDSC cells. We conclude that the spleen is an important contributor to tumor growth and metastases, and that splenectomy can blunt this effect by depletion of MDSC, changing the amount and characteristics of myeloid cells and enhancing activation of CTL.

The DC-HIL/Syndecan-4 Pathway Regulates Autoimmune Responses through Myeloid-Derived Suppressor Cells

Having discovered that the dendritic cell (DC)-associated heparan sulfate proteoglycan-dependent integrin ligand (DC-HIL) receptor on APCs inhibits T cell activation by binding to syndecan-4 (SD-4) on T cells, we hypothesized that the DC-HIL/SD-4 pathway may regulate autoimmune responses. Using experimental autoimmune encephalomyelitis (EAE) as a disease model, we noted an increase in SD-4(+) T cells in lymphoid organs of wild-type (WT) mice immunized for EAE. The autoimmune disease was also more severely induced (clinically, histologically, and immunophenotypically) in mice knocked out for SD-4 compared with WT cohorts. Moreover, infusion of SD-4(-/-) naive T cells during EAE induction into Rag2(-/-) mice also led to increased severity of EAE in these animals. Similar to SD-4 on T cells, DC-HIL expression was upregulated on myeloid cells during EAE induction, with CD11b(+)Gr-1(+) myeloid-derived suppressor cells (MDSCs) as the most expanded population and most potent T cell suppressor among the myeloid cells examined. The critical role of DC-HIL was supported by DC-HIL gene deletion or anti-DC-HIL treatment, which abrogated T cell suppressor activity of MDSCs, and also by DC-HIL activation inducing MDSC expression of IFN-γ, NO, and reactive oxygen species. Akin to SD-4(-/-) mice, DC-HIL(-/-) mice manifested exacerbated EAE. Adoptive transfer of MDSCs from EAE-affected WT mice into DC-HIL(-/-) mice reduced EAE severity to the level of EAE-immunized WT mice, an outcome that was precluded by depleting DC-HIL(+) cells from the infused MDSC preparation. Our findings indicate that the DC-HIL/SD-4 pathway regulates autoimmune responses by mediating the T cell suppressor function of MDSCs.

The Central Role of Arginine Catabolism in T-Cell Dysfunction and Increased Susceptibility to Infection After Physical Injury

To explore the hypothesis that decreased arginine availability by myeloid-derived suppressor cells (MDSCs) is a cause of T-cell dysfunction after physical injury (PI). Arginine is an essential amino acid for normal T-cell function whose availability becomes limited after PI. MDSCs expressing arginase 1 are induced by PI. T-cell dysfunction after PI seems to increase the risk of infection but the mechanisms that cause it are unclear. PI was created using a standard laparotomy model. Phenotypical and functional alterations in T cells were evaluated in vivo. MDSCs expressing arginase 1 were measured by flow cytometry. Infection after PI was created by intraperitoneal injection of Listeria monocytogenes. N-Hydroxy-Nor-L-arginine (Nor-NOHA) was used as an arginase inhibitor. The effect of arginine depletion on T-cell function and susceptibility to infection was assessed through adoptive transfer of MDSC or injection of arginase into noninjured mice. PI caused a decrease in intracellular arginine in T cells, loss of the T-cell receptor (TCR) CD3-ζ chain, inhibition of in vivo T-cell proliferation, memory, and cytotoxicity. PI exponentially increased bacterial growth and mortality to L. monocytogenes. T-cell dysfunction and increased infection were reversed by arginase inhibitor Nor-NOHA but were reproduced by adoptively transferring MDSC or injecting arginase 1 to noninjured mice. Arginine availability is decreased after PI coinciding with an induction of MDSC expressing arginase 1. Decreased arginine may inhibit T-cell function and increase susceptibility to infection after injury.

Abstract 87: The Accumulation of Myeloid-derived Suppressor Cells Is a Compensatory Response to the Development of Hypertension

The immune system plays a critical role in the development of hypertension. The immune response consists of pro-inflammatory cells, but also immunosuppressive cells that reduce T cell function. An important category of natural immunosuppressive cell is myeloid-derived suppressor cells (MDSC). We now show that blood and spleen CD11b+ Gr1+ myeloid cells are elevated 2-fold in both angiotensin II and L-NAME induced hypertension. These increased myeloid cells are MDSC in that they elevate IL-4R expression and suppress T cell proliferation. When hypertensive mice were depleted of MDSC, using either anti-Gr1 antibody or gemcitabine, there was a 15 mmHg rise in blood pressure and aggravation of T cells activation with increased production of IFN-γ, TNFα and IL-17 in both spleen and kidney. In contrast, adoptive transfer of MDSC reduced blood pressure in angiotensin-II induced hypertension by 25 mmHg (see Figure). These data suggest a new concept, that the accumulation of MDSC is a compensatory response to the inflammation induced by hypertension. They also indicate that MDSC play an important role in regulating blood pressure.

Protein Tyrosine Phosphatase 1B Deficiency Ameliorates Murine Experimental Colitis via the Expansion of Myeloid-Derived Suppressor Cells

Protein tyrosine phosphatase 1B (PTP1B) is a key molecule in modulating low-degree inflammatory conditions such as diabetes. The role of PTP1B in other chronic inflammations, however, remains unknown. Here, we report that PTP1B deficiency ameliorates Dextran Sulfate Sodium (DSS)-induced murine experimental colitis via expanding CD11b+Gr-1+ myeloid-derived suppressor cells (MDSCs). Employing DSS-induced murine experimental colitis as inflammatory animal model, we found that, compared with wild-type littermates, PTP1B-null mice demonstrated greater resistance to DSS-induced colitis, as reflected by slower weight-loss, greater survival rates and decreased PMN and macrophage infiltration into the colon. The evidence collectively also demonstrated that the resistance of PTP1B-null mice to DSS-induced colitis is based on the expansion of MDSCs. First, PTP1B-null mice exhibited a greater frequency of MDSCs in the bone marrow (BM), peripheral blood and spleen when compared with wild-type littermates. Second, PTP1B levels in BM leukocytes were significantly decreased after cells were induced into MDSCs by IL-6 and GM-CSF, and the MDSC induction occurred more rapidly in PTP1B-null mice than in wild-type littermates, suggesting PTP1B as a negative regulator of MDSCs. Third, the adoptive transfer of MDSCs into mice with DSS-colitis significantly attenuated colitis, which accompanies with a decreased serum IL-17 level. Finally, PTP1B deficiency increased the frequency of MDSCs from BM cells likely through enhancing the activities of signal transducer and activator of transcription 3 (STAT3) and Janus kinase 2 (JAK2). In conclusion, our study provides the first evidences that PTP1B deficiency ameliorates murine experimental colitis via expanding MDSCs.

Activated Invariant NKT Cells Control Central Nervous System Autoimmunity in a Mechanism That Involves Myeloid-Derived Suppressor Cells

Invariant NKT (iNKT) cells are a subset of T lymphocytes that recognize glycolipid Ags presented by the MHC class I-related protein CD1d. Activation of iNKT cells with glycolipid Ags, such as the marine sponge-derived reagent α-galactosylceramide (α-GalCer), results in the rapid production of a variety of cytokines and activation of many other immune cell types. These immunomodulatory properties of iNKT cells have been exploited for the development of immunotherapies against a variety of autoimmune and inflammatory diseases, but mechanisms by which activated iNKT cells confer disease protection have remained incompletely understood. In this study, we demonstrate that glycolipid-activated iNKT cells cooperate with myeloid-derived suppressor cells (MDSCs) in protecting mice against the development of experimental autoimmune encephalomyelitis (EAE) in mice, an animal model for multiple sclerosis. We show that α-GalCer induced the expansion and immunosuppressive activities of MDSCs in the spleen of mice induced for development of EAE. Disease protection in these animals also correlated with recruitment of MDSCs to the CNS. Depletion of MDSCs abrogated the protective effects of α-GalCer against EAE and, conversely, adoptive transfer of MDSCs from α-GalCer-treated mice ameliorated passive EAE induced in recipient animals. The cytokines GM-CSF, IL-4, and IFN-γ, produced by activated iNKT cells, and inducible NO synthase, arginase-1, and IL-10 produced by MDSCs, contributed to these effects. Our findings have revealed cooperative immunosuppressive interactions between iNKT cells and MDSCs that might be exploited for the development of improved immunotherapies for multiple sclerosis and other autoimmune and inflammatory diseases.

Myeloid-Derived Suppressor Cells (MDSCs) Induced In Vivo Following Activation of Invariant Natural Killer T (iNKT) Cells Prolong the Survival of Skin Allografts

Introduction: Transplantation is the most effective treatment for end-stage organ failure but long-term outcomes are hindered by chronic rejection and the side effects associated with prolonged immunosuppression. The induction of tolerance is an ideal solution to these problems and the contribution of immune cells other than T cells to this process is being increasingly recognized. In this study we investigated the potential impact of iNKT cells on the differentiation of myeloid-derived suppressor cells (MDSCs). We also examined the possibility of these in vivo expanded MDSCs to extend survival of an allogeneic skin transplant following adoptive transfer into a CD4+ TCR-transgenic (TCR-Tg) mouse model. Methods: B6 mice were injected i.v. with 1×106 TEa TCR-Tg CD4+ T cells (Va2+CD44-) on Day -1. To activate the TEa T cells these mice received 10x106 allogeneic B6xBALB/cF1 splenocytes i.v. on Day 0. Mice were also inoculated i.p. with either a-galactosylceramide (a-Gal) on Day 0 and 3 (to concomitantly activate endogenous iNKT cells) or PBS (as a control). On Day 5 Gr-1+CD11b+ MDSCs were flow sorted from the spleens of treated mice. Subsequently, B6 Rag-/- mice received 5x104 TEa T cells plus 5×104 sorted MDSCs from a-Gal or PBS treated mice. One day later all mice received an allogeneic B6xBALB/cF1 skin transplant. Results: As expected, transplanted mice that received TEa T cells alone rejected their grafts and those that received no cells accepted skin allografts long-term (median survival times (MST) 19.7 (n= 10) and >100 (n=10) days respectively). Mice that received a co-transfer of TEa T cells and MDSC from PBS treated mice rejected their grafts with a MST of 26.4 days (n=7). However, the addition of MDSC from a-Gal treated mice significantly extended graft survival compared to mice that received MDSC from PBS treated mice (MST=55 days (n=7), p=0.0002. Furthermore, flow sorted MDSC from a-Gal treated mice produced significantly more IFN-g and IL-10 compared to MDSC from PBS treated mice when stimulated in vitro (p=0.006 and p=0.0012 respectively). Conclusion: In summary, we have demonstrated that adoptive transfer of MDSCs, expanded in the presence of activated iNKT cells in vivo, significantly prolongs allograft survival in a mouse model of skin transplantation. We hypothesise that this extension in survival is IFN-g dependent as MDSC suppressive activity is lost in vivo and in vitro when IFN-g production is blocked.

Abstract 413: Involvement of myeloid derived suppressor cells (MDSC) in the changes of plasma amino acid profiles in cancer disease

Abstract Although maintained in healthy human, it is well known that plasma amino acid profiles are changed in various pathologic conditions, including liver disorder and cancer. We have previously reported the potential use of plasma amino acid profiling as diagnosis for several cancer disease. However, some questions remained for the mechanisms. The changes in plasma amino acid profiles in tumor disease were regulated by a lot of factors, including metabolic changes of tumors and immune cells. In this study, we focused on the role of immune cells, especially myeloid derived suppressor cells (MDSC). MDSC are characterized as immature myeloid cells that are precursors of dendritic cells, macrophages, and/or granulocytes, they are considered to accumulate in the blood, lymph nods, and at tumor sites in most patients and experimental animals with cancer and inhibit anti-tumor inflammation. Therefore we hypothesized that MDSC were involved in the changes in plasma amino acid profiles in tumor disease. To test our hypothesis, we employed C57BL/6 mice tumor model in which Lewis lung carcinoma (3LL) was injected subcutaneously. Using flow cytometry analysis, a significant percentage of MDSC were detected in spleens of tumor-bearing mice as compared to control normal mice. To assess the role of MDSC in the changes in plasma amino acid profile, we eliminated MDSC from the tumor-bearing mice by inducing apoptosis cell death on MDSC selectively in vivo. Flow cytometry results showed a significant decrease in the percentage of CD11b+ Gr1+ cells in splenocytes derived from MDSC deleted tumor-bearing mice as compared to tumor-bearing mice. To study the immunogenic effects of the MDSC deletion on the tumor-bearing mice, we performed histological analysis (H&amp;E staining) and extensive areas of inflammation within 3LL subcutaneous tumors were observed after the MDSC deletion. In addition, we investigated the effects of the MDSC deletion on the percentage of splenic T cell subpopulations by Flow cytometry. The percentage of CD4+ and CD8+ T cells were increased in spleens of MDSC deleted tumor-bearing mice compared to untreated tumor-bearing mice. To evaluate the effects of the MDSC depletion on the plasma amino acid profiles, plasma samples were collected from normal mice, tumor-bearing mice, and MDSC deleted tumor-bearing mice. Plasma amino acid concentration were measured by LC-MS. The MDSC deletion significantly changed the concentrations of some amino acids in plasma compared to the untreated tumor-bearing mice and normal mice. These changes in plasma amino acid concentrations were partially impeded by adoptive transfer of MDSC from tumor-bearing mice. Altogether, these results suggest that MDSC may be involved in the changes in plasma amino acid profiles in tumor disease. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 413. doi:10.1158/1538-7445.AM2011-413

Abstract B52: Characterizing the MDSCs phenotypes, expansion, and trafficking pattern and associated tumor-induced immune dysfunction

Abstract Among the prerequisites to be effective, immunotherapy must work in the presence of the host immunosuppression. Myeloid-derived suppressor cells (MDSCs) are one of the cellular mediators that have a role in limiting the induction of adaptive immune response and host responses to immunotherapy. MDSCs are an immature, heterogeneous cellular population, including hematopoietic precursors for granulocytes, macrophages, dendritic cells, and endothelial cells. They are infrequent in healthy hosts, but increase during tumor progression as a result of dysregulated myeloid cell expansion and differentiation. Tumor growth is frequently associated with an increased frequency of MDSCs that results in a loss in T-cell number and function through increased ROS and peroxynitrites production and arginase 1 (ARG1) and nitric oxide synthase 2 (NOS-2) upregulation. A better understanding of MDSCs expansion and the regulation of biodistribution and arrest would improve interventional strategies and cancer immunotherapy outcomes. Using five mammary tumor models, we have examined tumor-associated expansion of MDSCs and observed that this occurs in an organ-specific manner. Murine MDSCs can be subdivided into phenotypically and functionally distinct sub-populations according to Gr-1 expression levels; the CD11b+Gr-1 dim MDSCs with monocytic nuclei and strong T-cell suppressive activity, and CD11b+Gr-1bright having ring-shaped nuclei (polymorphonuclear cells) and a lower T-cell suppression profile. These cells can also be subset based on the expression of Ly-6C which is highly expressed on immature, bone marrow-derived cells and combined this Gr-1 expression Our studies revealed a significant increase in the Ly-6Cbright expression on the CD11b+Gr-1dim MDSCs in all organs studied in tumor-bearing mice as compared to non-tumor-bearing mice. In association with the increase in CD11b+Gr-1dimLy-6Cbright cells in the tumor non-parenchymal cells of 4T1 tumor bearing mice, increased transcripts levels of cytokines associated with MDSCs expansion (granulocyte-colony stimulating factor [G-CSF]) and MDSCs-associated immunosuppression (ARG-1, NOS-2, and cyclooxygenase 2 [COX-2]) while lower levels of these cytokines were observed in tumor NPCs from Cl66 tumor-bearing mice and non-tumor-bearing mice. In association with the increase in MDSCs and loss of T-cells in the 4T1 tumor-bearing mice there was a significant decrease in cytokine transcript levels that can enhance immune response (interferon gamma [IFN-γ] and interleukin 12 [IL-12]) from tumor non-parenchymal cells (NPCs) in 4T1 tumor bearing mice as compared to Cl66 tumor bearing mice and non-tumor bearing mice. Studies to assess where the MDSCs were expanding revealed that in 4T1 tumor bearing mice, frequency of the proliferating MDSCs is increased only in the spleen and liver compared to the non-tumor-bearing mice. Our studies also examined the affect of tumor burden on the trafficking of MDSCs. We observed that, after adoptive transfer of MDSCs isolated from 4T1 tumor-bearing mice, they arrest initially within the lungs in non-tumor-bearing mice. While in 4T1 tumor-bearing mice they rapidly transverse the lungs with arrest within the tumor tissue. We and others reported that the increase in MDSCs frequencies is associated with a decrease in CD3+ cells function and numbers. We observed a decrease in frequency of all T-cell subsets in the thymus of tumor bearing mice tested compared to the non-tumor-bearing mice suggesting defects in T-cell maturation associated with tumor burden. We investigated the effect of tumor burden on the phenotype of the CD3+ cells using the Annexin V staining and found that 4T1 tumor bearing mice have increased frequency of Annexin V positive CD3+ T-cells that are infiltrating tumor tissue compared to those in other organs. Citation Information: Clin Cancer Res 2010;16(14 Suppl):B52.