Gr‐1+CD11b+ myeloid cells efficiently home to site of injury after intravenous administration and enhance diabetic wound healing by neoangiogenesis

Vascularization is an important factor that affects diabetic wound healing. There is increasing evidence that myeloid cell lineages play a role in neovascularization. In this study, the efficiency of Gr‐1+CD11b+ myeloid cells to home to the site of injury and enhance diabetic wound healing by neoangiogenesis after intravenous administration was investigated. Gr‐1+CD11b+ myeloid cells were injected into tail vein after establishment of dorsal window chamber, hindlimb ischaemia and ear‐punch injury in diabetic or non‐diabetic mice. The Gr‐1+CD11b+ myeloid cells efficiently homed to the site of injury after intravenous administration and increased neoangiogenesis. The chemokine receptor type 4 (CXCR4) is robustly expressed by Gr‐1+CD11b+ myeloid cells. Inhibition of CXCR4 decreases the homing ability of Gr‐1+CD11b+ myeloid cells to the site of injury, which indicates that the CXCR4/SDF‐1 axis plays an important role in the homing of Gr‐1+CD11b+ myeloid cells to the site of injury. In addition, Gr‐1+CD11b+ myeloid cells were found to improve blood flow recovery of ischaemic limb and enhance wound healing in diabetic mice by neoangiogenesis after intravenous administration. Taken together, the results of this study suggest that Gr‐1+CD11b+ myeloid cells may serve as a potential cell therapy for diabetic wound healing.     

Bone marrow derived cells in tumor angiogenesis and growth: are they the good,the bad or the evil?

Increasing evidence implicates an important role for a variety of bone marrow derived cells (BMDCs) in tumor angiogenesis and metastatic tumor growth. These cells are derived either from the hematopoietic or mesenchymal cell lineage, and they are distinguished, in part, by the expression of the panhematopoietic marker ‐ CD45. Some of these cell populations can colonize tumors perivascularily, and appear to promote angiogenesis and tumor cell proliferation by paracraine mechanisms, whereas others can contribute “directly” to the growth of tumor vessel capillaries or metastases. In this review we focus in particular on the role of hemangiocytes or recruited bone marrow derived circulating cells (RBCCs) in neovascularization, the contribution of VEGFR1+ hematopoietic stem cells and endothelial precursor cells in metastasis, and the involvement of myeloid derived suppressor CD11b+/Gr‐1+ cells in the resistance of tumors to certain antiangiogenic drugs, e.g., VEGF blocking antibodies.     

Expression of the B-Cell Receptor Component CD79a on Immature Myeloid Cells Contributes to Their Tumor Promoting Effects

The role of myeloid derived suppressor cells (MDSCs) in promoting tumorigenesis is well-established, and significant effort is being made to further characterize surface markers on MDSCs both for better diagnosis and as potential targets for therapy. Here we show that the B cell receptor adaptor molecule CD79a is unexpectedly expressed on immature bone marrow myeloid cells, and is upregulated on MDSCs generated in multiple different mouse models of metastatic but not non-metastatic cancer. CD79a on MDSCs is upregulated and activated in response to soluble factors secreted by tumor cells. Activation of CD79a on mouse MDSCs, by crosslinking with a specific antibody, maintained their immature phenotype (CD11b+Gr1+), enhanced their migration, increased their suppressive effect on T cell proliferation, and increased secretion of pro-tumorigenic cytokines such as IL-6 and CCL22. Furthermore, crosslinking CD79a on myeloid cells activated signaling through Syk, BLNK, ERK and STAT3 phosphorylation. In vivo, CD79+ myeloid cells showed enhanced ability to promote primary tumor growth and metastasis. Finally we demonstrate that CD79a is upregulated on circulating myeloid cells from lung cancer patients, and that CD79a+ myeloid cells infiltrate human breast tumors. We propose that CD79a plays a functional role in the tumor promoting effects of myeloid cells, and may represent a novel target for cancer therapy.     

Stromal Cell-Derived Factor-1/CXCL12 Contributes to MMTV-Wnt1 Tumor Growth Involving Gr1+CD11b+ Cells

Background

Histological examinations of MMTV-Wnt1 tumors reveal drastic differences in the tumor vasculature when compared to MMTV-Her2 tumors. However, these differences have not been formally described, nor have any angiogenic factors been implicated to be involved in the Wnt1 tumors.

Methodology/Principal Findings

Here, we show that MMTV-Wnt1 tumors were more vascularized than MMTV-Her2 tumors, and this correlated with significantly higher expression of a CXC chemokine, stromal cell-derived factor-1 (SDF1/CXCL12) but not with VEGFA. Isolation of various cell types from Wnt1 tumors revealed that SDF1 was produced by both tumor myoepithelial cells and stromal cells, whereas Her2 tumors lacked myoepithelial cells and contained significantly less stroma. The growth of Wnt1 tumors, but not Her2 tumors, was inhibited by a neutralizing antibody to SDF1, but not by neutralization of VEGFA. Anti-SDF1 treatment decreased the proportion of infiltrating Gr1⁺ myeloid cells in the Wnt1 tumors, which correlated with a decrease in the percentage of endothelial cells. The involvement of Gr1⁺ cells was evident from the retardation of Wnt1 tumor growth following in vivo depletion of these cells with an anti-Gr1-specific antibody. This degree of inhibition on Wnt1 tumor growth was comparable, but not additive, to the effect observed with anti-SDF1, indicative of overlapping mechanisms of inhibition. In contrast, Her2 tumors were not affected by the depletion of Gr1⁺ cells.

Conclusions/Significance

We demonstrated that SDF1 is important for Wnt1, but not for HER2, in inducing murine mammary tumor and the role of SDF1 in tumorigenesis involves Gr1⁺ myeloid cells to facilitate growth and/or angiogenesis.     

The role of Gr1+ cells after anti-CD20 treatment in type 1 diabetes in nonobese diabetic mice

Studies suggest that Gr1(+)CD11b(+) cells have immunoregulatory function, and these cells may play an important role in autoimmune diseases. In this study, we investigated the regulatory role of Gr1(+)CD11b(+) cells in protecting against type 1 diabetes in NOD mice. In this study, we showed that temporary B cell depletion induced the expansion of Gr1(+)CD11b(+) cells. Gr1(+)CD11b(+) cells not only directly suppress diabetogenic T cell function but also can induce regulatory T cell differentiation in a TGF-β-dependent manner. Furthermore, we found that Gr1(+)CD11b(+) cells could suppress diabetogenic CD4 and CD8 T cell function in an IL-10-, NO-, and cell contact-dependent manner. Interestingly, single anti-Gr1 mAb treatment can also induce a transient expansion of Gr1(+)CD11b(+) cells that delayed diabetes development in NOD mice. Our data suggest that Gr1(+)CD11b(+) cells contribute to the establishment of immune tolerance to pancreatic islet autoimmunity. Manipulation of Gr1(+)CD11b(+) cells could be considered as a novel immunotherapy for the prevention of type 1 diabetes.     

Melanoma cell expression of CD200 inhibits tumor formation and lung metastasis via inhibition of myeloid cell functions

CD200 is a cell surface glycoprotein that functions through engaging CD200 receptor on cells of the myeloid lineage and inhibits their functions. Expression of CD200 has been implicated in a variety of human cancer cells including melanoma cells and has been thought to play a protumor role. To investigate the role of cancer cell expression of CD200 in tumor formation and metastasis, we generated CD200-positive and CD200-negative B16 melanoma cells. Subcutaneous injection of CD200-positive B16 melanoma cells inhibited tumor formation and growth in C57BL/6 mice but not in Rag1^−/−C57BL/6 mice. However, i.v. injection of CD200-positive B16 melanoma cells dramatically inhibited tumor foci formation in the lungs of both C57BL/6 and Rag1^−/−C57BL6 mice. Flow cytometry analysis revealed higher expression of CD200R in Gr1⁺ myeloid cells in the lung than in peripheral myeloid cells. Depletion of Gr1⁺ cells or stimulation of CD200R with an agonistic antibody in vivo dramatically inhibited tumor foci formation in the lungs. In addition, treatment with tumor antigen specific CD4 or CD8 T cells or their combination yielded a survival advantage for CD200 positive tumor bearing mice over mice bearing CD200-negative tumors. Taken together, we have revealed a novel role for CD200-CD200R interaction in inhibiting tumor formation and metastasis. Targeting CD200R may represent a novel approach for cancer immunotherapy.     

Oxidative stress regulates expression of VEGFR1 in myeloid cells: link to tumor-induced immune suppression in renal cell carcinoma

Metastatic renal cell carcinoma (RCC) associates with overproduction of vascular endothelial growth factor (VEGF) due to the mutation/inactivation of the von Hippel-Lindau (VHL) tumor suppressor gene. Herein we demonstrate that implantation of human RCC tumor cells into athymic nude mice promotes the appearance of VEGF receptor 1 (VEGFR1)/CD11b double-positive myeloid cells in peripheral blood. Avastin-mediated VEGF neutralization was capable of significantly reducing the numbers of circulating VEGFR1+ myeloid cells. Conversely, up-regulation of VEGFR1 by myeloid cells could also be achieved in vitro by coculturing bone marrow cells with RCC-conditioned medium or by short-term exposure of naive myeloid cells to oxidative stress. Treatment of myeloid cells with H2O2, lipid peroxidation product 4-hydroxy-2(E)-nonenal, or an inhibitor of thioredoxin reductase all resulted in increased expression of VEGFR1. Furthermore, after exposure to oxidative stress, myeloid cells acquire immunosuppressive features and become capable of inhibiting T cell proliferation. Data suggest that tumor-induced oxidative stress may promote both VEGFR1 up-regulation and immunosuppressive function in bone marrow-derived myeloid cells. Analysis of tumor tissue and peripheral blood from patients with metastatic RCC revealed that VEGFR1+ cells can be also found in cancer patients. Restoration of immunocompetence in metastatic RCC patients by pharmacological elimination of VEGFR1+ cells may have a significant impact on the therapeutic efficacy of cancer vaccines or other immune-based therapies.     

Adenosinergic regulation of the expansion and immunosuppressive activity of CD11b+Gr1+ cells

Extracellular adenosine and purine nucleotides are elevated in many pathological situations associated with the expansion of CD11b(+)Gr1(+) myeloid-derived suppressor cells (MDSCs). Therefore, we tested whether adenosinergic pathways play a role in MDSC expansion and functions. We found that A(2B) adenosine receptors on hematopoietic cells play an important role in accumulation of intratumoral CD11b(+)Gr1(high) cells in a mouse Lewis lung carcinoma model in vivo and demonstrated that these receptors promote preferential expansion of the granulocytic CD11b(+)Gr1(high) subset of MDSCs in vitro. Flow cytometry analysis of MDSCs generated from mouse hematopoietic progenitor cells revealed that the CD11b(+)Gr-1(high) subset had the highest levels of CD73 (ecto-5'-nucleotidase) expression (Δmean fluorescence intensity [MFI] of 118.5 ± 16.8), followed by CD11b(+)Gr-1(int) (ΔMFI of 57.9 ± 6.8) and CD11b(+)Gr-1(-/low) (ΔMFI of 12.4 ± 1.0) subsets. Even lower levels of CD73 expression were found on Lewis lung carcinoma tumor cells (ΔMFI of 3.2 ± 0.2). The high levels of CD73 expression in granulocytic CD11b(+)Gr-1(high) cells correlated with high levels of ecto-5'-nucleotidase enzymatic activity. We further demonstrated that the ability of granulocytic MDSCs to suppress CD3/CD28-induced T cell proliferation was significantly facilitated in the presence of the ecto-5'-nucleotidase substrate 5'-AMP. We propose that generation of adenosine by CD73 expressed at high levels on granulocytic MDSCs may promote their expansion and facilitate their immunosuppressive activity.     

A role for immature myeloid cells in immune senescence

The reduced efficiency of the mammalian immune system with aging increases host susceptibility to infectious and autoimmune diseases. However, the mechanisms responsible for these pathologic changes are not well understood. In this study, we demonstrate that the bone marrow, blood, and secondary lymphoid organs of healthy aged mice possess increased numbers of immature myeloid cells that are phenotypically similar to myeloid-derived suppressor cells found in lymphoid organs of mice with progressive tumors and other pathologic conditions associated with chronic inflammation. These cells are characterized by the presence of Gr1 and CD11b markers on their surfaces. Gr1(+)CD11b(+) cells isolated from aged mice possess an ability to suppress T cell proliferation/activation and produce heightened levels of proinflammatory cytokines, both constitutively and upon activation, including IL-12, which promotes an excessive production of IFN-γ. IFN-γ priming is essential for excessive proinflammatory cytokine production and the suppressive activities by Gr1(+)CD11b(+) cells from aged mice. These cells suppress T cell proliferation through an NO-dependent mechanism, as depletion of splenic Gr1(+) cells reduces NO levels and restores T cell proliferation. Insights into mechanisms responsible for the proinflammatory and immune suppressive activities of Gr1(+)CD11b(+) cells from aged mice have uncovered a defective PI3K-Akt signaling pathway, leading to a reduced Akt-dependent inactivation of GSK3β. Our data demonstrate that abnormal activities of the Gr1(+)CD11b(+) myeloid cell population from aged mice could play a significant role in the mechanisms responsible for immune senescence.     

CTL induction of tumoricidal nitric oxide production by intratumoral macrophages is critical for tumor elimination

To characterize mechanisms of CTL inhibition within an ocular tumor microenvironment, tumor-specific CTLs were transferred into mice with tumors developing within the anterior chamber of the eye or skin. Ocular tumors were resistant to CTL transfer therapy whereas skin tumors were sensitive. CTLs infiltrated ocular tumors at higher CTL/tumor ratios than in skin tumors and demonstrated comparable ex vivo effector function to CTLs within skin tumors indicating that ocular tumor progression was not due to decreased CTL accumulation or inhibited CTL function within the eye. CD11b(+)Gr-1(+)F4/80(-) cells predominated within ocular tumors, whereas skin tumors were primarily infiltrated by CD11b(+)Gr-1(-)F4/80(+) macrophages (Ms), suggesting that myeloid derived suppressor cells may contribute to ocular tumor growth. However, CD11b(+) myeloid cells isolated from either tumor site suppressed CTL activity in vitro via NO production. Paradoxically, the regression of skin tumors by CTL transfer therapy required NO production by intratumoral Ms indicating that NO-producing intratumoral myeloid cells did not suppress the effector phase of CTL. Upon CTL transfer, tumoricidal concentrations of NO were only produced by skin tumor-associated Ms though ocular tumor-associated Ms demonstrated comparable expression of inducible NO synthase protein suggesting that NO synthase enzymatic activity was compromised within the eye. Correspondingly, in vitro-activated Ms limited tumor growth when co-injected with tumor cells in the skin but not in the eye. In conclusion, the decreased capacity of Ms to produce NO within the ocular microenvironment limits CTL tumoricidal activity allowing ocular tumors to progress.     

CD11b+/Gr-1+ immature myeloid cells mediate suppression of T cells in mice bearing tumors of IL-1beta-secreting cells

Tumor cells secreting IL-1beta are invasive and metastatic, more than the parental line or control mock-transfected cells, and concomitantly induce in mice general immune suppression of T cell responses. Suppression strongly correlates with accumulation in the peripheral blood and spleen of CD11b+/Gr-1+ immature myeloid cells and hematological alterations, such as splenomegaly, leukocytosis, and anemia. Resection of large tumors of IL-1beta-secreting cells restored immune reactivity and hematological alterations within 7-10 days. Treatment of tumor-bearing mice with the physiological inhibitor of IL-1, the IL-1R antagonist, reduced tumor growth and attenuated the hematological alterations. Depletion of CD11b+/Gr-1+ immature myeloid cells from splenocytes of tumor-bearing mice abrogated suppression. Despite tumor-mediated suppression, resection of large tumors of IL-1beta-secreting cells, followed by a challenge with the wild-type parental cells, induced resistance in mice; protection was not observed in mice bearing tumors of mock-transfected fibrosarcoma cells. Altogether, we show in this study that tumor-derived IL-1beta, in addition to its proinflammatory effects on tumor invasiveness, induces in the host hematological alterations and tumor-mediated suppression. Furthermore, the antitumor effectiveness of the IL-1R antagonist was also shown to encompass restoration of hematological alterations, in addition to its favorable effects on tumor invasiveness and angiogenesis that have previously been described by us.     

Histamine deficiency promotes inflammation-associated carcinogenesis through reduced myeloid maturation and accumulation of CD11b+Ly6G+ immature myeloid cells

Histidine decarboxylase (HDC), the unique enzyme responsible for histamine generation, is highly expressed in myeloid cells, but its function in these cells is poorly understood. Here we show that Hdc-knockout mice show a high rate of colon and skin carcinogenesis. Using Hdc-EGFP bacterial artificial chromosome (BAC) transgenic mice in which EGFP expression is controlled by the Hdc promoter, we show that Hdc is expressed primarily in CD11b(+)Ly6G(+) immature myeloid cells (IMCs) that are recruited early on in chemical carcinogenesis. Transplant of Hdc-deficient bone marrow to wild-type recipients results in increased CD11b(+)Ly6G(+) cell mobilization and reproduces the cancer susceptibility phenotype of Hdc-knockout mice. In addition, Hdc-deficient IMCs promote the growth of tumor allografts, whereas mouse CT26 colon cancer cells downregulate Hdc expression through promoter hypermethylation and inhibit myeloid cell maturation. Exogenous histamine induces the differentiation of IMCs and suppresses their ability to support the growth of tumor allografts. These data indicate key roles for Hdc and histamine in myeloid cell differentiation and CD11b(+)Ly6G(+) IMCs in early cancer development.     

Cytokine Levels Correlate with Immune Cell Infiltration after Anti-VEGF Therapy in Preclinical Mouse Models of Breast Cancer

The effect of blocking VEGF activity in solid tumors extends beyond inhibition of angiogenesis. However, no studies have compared the effectiveness of mechanistically different anti-VEGF inhibitors with respect to changes in tumor growth and alterations in the tumor microenvironment. In this study we use three distinct breast cancer models, a MDA-MB-231 xenograft model, a 4T1 syngenic model, and a transgenic model using MMTV-PyMT mice, to explore the effects of various anti-VEGF therapies on tumor vasculature, immune cell infiltration, and cytokine levels. Tumor vasculature and immune cell infiltration were evaluated using immunohistochemistry. Cytokine levels were evaluated using ELISA and electrochemiluminescence. We found that blocking the activation of VEGF receptor resulted in changes in intra-tumoral cytokine levels, specifically IL-1β, IL-6 and CXCL1. Modulation of the level these cytokines is important for controlling immune cell infiltration and ultimately tumor growth. Furthermore, we demonstrate that selective inhibition of VEGF binding to VEGFR2 with r84 is more effective at controlling tumor growth and inhibiting the infiltration of suppressive immune cells (MDSC, Treg, macrophages) while increasing the mature dendritic cell fraction than other anti-VEGF strategies. In addition, we found that changes in serum IL-1β and IL-6 levels correlated with response to therapy, identifying two possible biomarkers for assessing the effectiveness of anti-VEGF therapy in breast cancer patients.     

mTOR inhibitor INK128 attenuates systemic lupus erythematosus by regulating inflammation-induced CD11b+Gr1+ cells

Systemic lupus erythematosus (SLE) is an autoimmune disease, characterized by systemic chronic inflammation that can affect multiple major organ systems. Although the etiology of SLE is known to involve a variety of factors such as the environment, random factors and genetic susceptibility, the exact role of CD11b⁺Gr1⁺ myeloid cells in lupus progression is not fully understood. Myeloid-derived CD11b⁺Gr1⁺ cells are thought to be a heterogeneous group of immature myeloid cells with immune function. Some studies have reported that CD11b⁺Gr1⁺ cells and the activation of mTOR pathway are involved in the pathogenesis of systemic lupus erythematosus (SLE). However, it is still not clarified about the mechanism of influence of lupus microenvironment and mTOR signaling on CD11b⁺Gr1⁺ cells. In the present study, we found that the percentage of CD11b⁺Gr1⁺ cells increased prior to the abnormal changes of Th17, Treg, T and B cells during lupus development. TLR7 and IFN-α signaling synergized to promote CD11b⁺Gr1⁺ cell accumulation in an mTOR-dependent manner. Moreover, compared to a traditional mTOR inhibitor, INK128 inhibited more effectively the disease activity via regulating CD11b⁺Gr1⁺ cell expansion and functions. Furthermore, TLR7/IFN-α-modified CD11b⁺Gr1⁺ cells promoted unbalance of Th17/Tregs and were inclined to differentiate into macrophages via the mTOR pathway. In conclusion, CD11b⁺Gr1⁺ cells increased in the early stages of the lupus progression and mTOR pathway was critical for CD11b⁺Gr1⁺ cells in lupus development, suggesting the changes of inflammation-induced CD11b⁺Gr1⁺ cells initate lupus development. We also provide evidence for the first time that INK128, a second generation mTOR inhibitor, has a good therapeutic action on lupus development by regulating CD11b⁺Gr1⁺ cells.     

S100A8/A9 activate key genes and pathways in colon tumor progression

The tumor microenvironment plays an important role in modulating tumor progression. Earlier, we showed that S100A8/A9 proteins secreted by myeloid-derived suppressor cells (MDSC) present within tumors and metastatic sites promote an autocrine pathway for accumulation of MDSC. In a mouse model of colitis-associated colon cancer, we also showed that S100A8/A9-positive cells accumulate in all regions of dysplasia and adenoma. Here we present evidence that S100A8/A9 interact with RAGE and carboxylated glycans on colon tumor cells and promote activation of MAPK and NF-κB signaling pathways. Comparison of gene expression profiles of S100A8/A9-activated colon tumor cells versus unactivated cells led us to identify a small cohort of genes upregulated in activated cells, including Cxcl1, Ccl5 and Ccl7, Slc39a10, Lcn2, Zc3h12a, Enpp2, and other genes, whose products promote leukocyte recruitment, angiogenesis, tumor migration, wound healing, and formation of premetastatic niches in distal metastatic organs. Consistent with this observation, in murine colon tumor models we found that chemokines were upregulated in tumors, and elevated in sera of tumor-bearing wild-type mice. Mice lacking S100A9 showed significantly reduced tumor incidence, growth and metastasis, reduced chemokine levels, and reduced infiltration of CD11b(+)Gr1(+) cells within tumors and premetastatic organs. Studies using bone marrow chimeric mice revealed that S100A8/A9 expression on myeloid cells is essential for development of colon tumors. Our results thus reveal a novel role for myeloid-derived S100A8/A9 in activating specific downstream genes associated with tumorigenesis and in promoting tumor growth and metastasis.     

Marked expansion of CD11c+CD8+ T-cells in melanoma-bearing mice induced by anti-4-1BB monoclonal antibody

4-1BB (CD137), a member of the tumor necrosis factor receptor superfamily, is expressed on activated T-cells, and 4-1BB signaling due to interaction with 4-1BB ligand or ligation with anti-4-1BB monoclonal antibody (mAb) costimulates T cells. It has been shown that administration of anti-4-1BB mAb induces anti-tumor immunity in mice, but the nature of the cellular subsets responsible for this immunity is uncertain. In this study we found that anti-4-1BB mAb administration to B16F10 melanoma-bearing mice induced marked expansion of CD11c+CD8+ T-cells in parallel with suppression of pulmonary tumors. The mAb-treated mice produced higher levels of IFN- in their tumor tissues, spleen and lymph nodes than mice exposed to control antibody. When the CD11c+CD8+ T-cells were purified and re-stimulated in vitro, they produced high levels of the Th1 cytokines, IFN- and IL-2, but low levels of the Th2 cytokines, IL-4 and IL-10. Furthermore, they expressed high levels of 4-1BB and CD107a, a marker of activated cytotoxic T-lymphocytes. Our results suggest that CD11c+CD8+ T-cells play a role in the anti-tumor immunity induced by anti-4-1BB mAb.     

Identification and characterization of novel bone marrow myeloid DEC205+Gr-1+ cell subsets that differentially express chemokine and TLRs

Bone marrow-derived immunomodulatory cytokines impart a critical function in the regulation of innate immune responses and hemopoiesis. However, the source of immunomodulatory cytokines in murine bone marrow and the cellular immune mechanisms that control local cytokine secretion remain poorly defined. Herein, we identified a population of resident murine bone marrow myeloid DEC205(+)CD11c(-)B220(-)Gr1(+)CD8alpha(-)CD11b(+) cells that respond to TLR2, TLR4, TLR7, TLR8, and TLR9 agonists as measured by the secretion of proinflammatory and anti-inflammatory cytokines in vitro. Phenotypic and functional analyses revealed that DEC205(+)CD11b(+)Gr-1(+) bone marrow cells consist of heterogeneous populations of myeloid cells that can be divided into two main cell subsets based on chemokine and TLR gene expression profile. The DEC205(+)CD11b(+)Gr-1(low) cell subset expresses high levels of TLR7 and TLR9 and was the predominant source of IL-6, TNF-alpha, and IL-12 p70 production following stimulation with the TLR7 and TLR9 agonists CpG and R848, respectively. In contrast, the DEC205(+)CD11b(+)Gr-1(high) cell subset did not respond to CpG and R848 stimulation, which correlated with their lack of TLR7 and TLR9 expression. Similarly, a differential chemokine receptor expression profile was observed with higher expression of CCR1 and CXCR2 found in the DEC205(+)CD11(+)Gr-1(high) cell subset. Thus, we identified a previously uncharacterized population of resident bone marrow cells that may be implicated in the regulation of local immune responses in the bone marrow.     

Generation of antigen-presenting cells from tumor-infiltrated CD11b myeloid cells with DNA demethylating agent 5-aza-2′-deoxycytidine

Tumor-recruited CD11b myeloid cells, including myeloid-derived suppressor cells, play a significant role in tumor progression, as these cells are involved in tumor-induced immune suppression and tumor neovasculogenesis. On the other hand, the tumor-infiltrated CD11b myeloid cells could potentially be a source of immunostimulatory antigen-presenting cells (APCs), since most of these cells represent common precursors of both dendritic cells and macrophages. Here, we investigated the possibility of generating mature APCs from tumor-infiltrated CD11b myeloid cells. We demonstrate that in vitro exposure of freshly excised mouse tumors to DNA methyltransferase inhibitor 5-aza-2′-deoxycytidine (decitabine, AZA) results in selective elimination of tumor cells, but, surprisingly it also enriches CD45⁺ tumor-infiltrated cells. The majority of “post-AZA” surviving CD45⁺ tumor-infiltrated cells were represented by CD11b myeloid cells. A culture of isolated tumor-infiltrated CD11b cells in the presence of AZA and GM-CSF promoted their differentiation into mature F4/80/CD11c/MHC class II-positive APCs. These tumor-derived myeloid APCs produced substantially reduced amounts of immunosuppressive (IL-13, IL-10, PGE₂), pro-angiogenic (VEGF, MMP-9) and pro-inflammatory (IL-1beta, IL-6, MIP-2) mediators than their precursors, freshly isolated tumor-infiltrated CD11b cells. Vaccinating naïve mice with ex vivo generated tumor-derived APCs resulted in the protection of 70% mice from tumor outgrowth. Importantly, no loading of tumor-derived APC with exogenous antigen was needed to stimulate T cell response and induce the anti-tumor effect. Collectively, our results for the first time demonstrate that tumor-infiltrated CD11b myeloid cells can be enriched and differentiated in the presence of DNA demethylating agent 5-aza-2′-deoxycytidine into mature tumor-derived APCs, which could be used for cancer immunotherapy.     

Salmonella-mediated tumor regression involves targeting of tumor myeloid suppressor cells causing a shift to M1-like phenotype and reduction in suppressive capacity

The effectiveness of attenuated Salmonella in inhibiting tumor growth has been demonstrated in many therapeutic models, but the precise mechanisms remain incompletely understood. In this study, we show that the anti-tumor capacity of Salmonella depends on a functional MyD88-TLR pathway and is independent of adaptive immune responses. Since myeloid suppressor cells play a critical role in tumor growth, we investigated the consequences of Salmonella treatment on myeloid cell recruitment, phenotypic characteristics, and functional activation in spleen and tumor tissue of B16.F1 melanoma-bearing mice. Salmonella treatment led to increased accumulation of splenic and intratumoral CD11b⁺Gr-1⁺ myeloid cells, exhibiting significantly increased expression of various activation markers such as MHC class II, costimulatory molecules, and Sca-1/Ly6A proteins. Gene expression analysis showed that Salmonella treatment induced expression of iNOS, arginase-1 (ARG1), and IFN-γ in the spleen, but down-regulated IL-4 and TGF-β. Within the tumor, expression of iNOS, IFN-γ, and S100A9 was markedly increased, but ARG1, IL-4, TGF-β, and VEGF were inhibited. Functionally, splenic CD11b⁺ cells maintained their suppressive capacity following Salmonella treatment, but intratumoral myeloid cells had significantly reduced suppressive capacity. Our findings demonstrate that administration of attenuated Salmonella leads to phenotypic and functional maturation of intratumoral myeloid cells making them less suppressive and hence enhancing the host’s anti-tumor immune response. Modalities that inhibit myeloid suppressor cells may be useful adjuncts in cancer immunotherapy.