Potent Suppressive Activity Research Articles

Plasticity of myeloid-derived suppressor cells in cancer

In recent years, myeloid-derived suppressor cells (MDSC) have emerged as one of the major inhibitors of immune effector cell function in cancer. MDSC represent a heterogeneous population of largely immature myeloid cells that are characterized by a pathological state of activation and display potent immune suppressive activity. Two major subsets of MDSC have been identified: monocytic (M-MDSC) and polymorphonuclear (PMN-MDSC). PMN-MSDC share phenotypic and morphologic features with neutrophils, whereas M-MDSC are similar to monocytes and are characterized by high plasticity. Differentiation of M-MDSC to macrophages and dendritic cells is shaped by tumor microenvironment. In recent years, the mechanisms of this process start to emerge.

Discovery of 18β-glycyrrhetinic acid conjugated aminobenzothiazole derivatives as Hsp90-Cdc37 interaction disruptors that inhibit cell migration and reverse drug resistance

A series of 18β-glycyrrhetinic acid (GA) conjugated aminobenzothiazole derivatives were designed, synthesized and evaluated for disruption activity of Hsp90-Cdc37 as well as the effects of in vitro cell migration. These compounds exhibited relatively good disruption activity against Hsp90-Cdc37 with IC50 values in low micromolar range. A docking study of the most active compound 11g revealed key interactions between 11g and Hsp90-Cdc37 complex in which the benzothiazole moiety and the amine chain group were important for improving activity. It is noteworthy that further antitumor activity screening revealed that some compounds exhibited better inhibitory activity than the commercial anticancer drug 5-FU and showed potent suppression activity against drug-resistant cancer cells. In particular, compound 11 g appeared to be the most potent compound against the A549 cell line, at least partly, by inhibition of the activity of Hsp90 and apoptosis induction. The treatment of A549 cells with compound 11g resulted in inhibition of in vitro cell migration through wound healing assay and S phase of cell cycle arrested. In addition, 11g-induced apoptosis was significantly facilitated in A549 cells. Thus, we conclude that GA aminobenzothiazole derivatives may be the potential Hsp90-Cdc37 disruptors with the ability to suppress cells migration and reversed drug-resistant.

Transitory presence of myeloid-derived suppressor cells in neonates is critical for control of inflammation.

Myeloid-derived suppressor cells (MDSC) are pathologically activated and relatively immature myeloid cells, which are implicated in the immune regulation of many pathologic conditions1,2. Phenotypically and morphologically MDSC are similar to neutrophils (PMN-MDSC) and monocytes (M-MDSC). However, they have potent suppressive activity, a distinct gene expression profile, and biochemical characteristics3. None or very few MDSC are observed in steady state physiological conditions. Therefore, until recently, accumulation of MDSC was considered as a consequence of pathological process or pregnancy. Here, we report that MDSC with a potent ability to suppress T cells are present during the first weeks of life in mice and humans. MDSC suppressive activity was triggered by lactoferrin and mediated by nitric oxide, PGE2, and S100A9/A8 proteins. Newborn MDSC had a transcriptome similar to that of tumor MDSC, but with a strong up-regulation of an antimicrobial gene network and had potent antibacterial activity. MDSC played a critical role in control of experimental necrotizing enterocolitis (NEC) in newborn mice. MDSC in infants with very low-weight, which are prone to the development of NEC, had lower MDSC levels and suppressive activity than infants with normal weight. Thus, the transitory presence of MDSC may be critical for regulation of inflammation in newborns.

C-C Chemokine Receptor Type 2-Dependent Migration of Myeloid-Derived Suppressor Cells in Protection of Islet Transplants.

Islet transplantation is a promising therapeutic approach to restore the physical response to blood glucose in type 1 diabetes. Current chronic use of immunosuppressive reagents for preventing islet allograft rejection is associated with severe complications. In addition, many of the immunosuppressive drugs are diabetogenic. The induction of transplant tolerance to eliminate the dependency on immunosuppression is ideal, but remains challenging. Addition of hepatic stellate cells allowed generation of myeloid-derived suppressor cells (MDSC) from precursors in mouse bone marrow. Migration of MDSC was examined in an islet allograft transplant model by tracking the systemic administered MDSC from CD45.1 congenic mice. The generated MDSC were expressed C-C chemokine receptor type 2 (CCR2), which was enhanced by exposure to interferon-γ. A single systemic administration of MDSC markedly prolonged survival of islet allografts without requirement of immunosuppression. Tracking the administered MDSC showed that they promptly migrated to the islet graft sites, at which point they exerted potent immune suppressive activity by inhibiting CD8 T cells, enhancing regulatory T cell activity. MDSC generated from CCR2 mice failed to be mobilized and lost tolerogenic activity in vivo, but sustained suppressive activity in vitro. MDSC migration was dependent on expression of CCR2, whereas CCR2 does not directly participate in immune suppression. Expression of CCR2 needs to be closely monitored for quality control purpose when MDSC are generated in vitro for immune therapy.

Pharmacokinetic/pharmacodynamic drug evaluation of benralizumab for the treatment of asthma

ABSTRACTIntroduction: In many severe asthmatics, eosinophils cause inflammation and airways hyperresponsiveness, resulting in frequent exacerbations, impaired lung function, and reduced quality of life. Interleukin-5 (IL-5) is a key cytokine for eosinophil growth, differentiation, recruitment, activation, and survival. Anti-IL-5-based therapies (mepolizumab and reslizumab are humanized monoclonal antibodies (hmAbs) that recognize free IL-5, benralizumab is a hmAb directed at the α subunit of the IL-5R) target the IL-5-signaling in eosinophilic asthma.Areas covered: The pharmacodynamic/pharmacokinetic profile of benralizumab and how it provided indications that permitted optimization of the design and timelines of the pivotal trials are described.Expert opinion: Benralizumab has the advantage over other anti-IL-5 therapies to target the IL-5Rα itself. Afucosylation enhances its interaction with its binding site and facilitates its pharmacological activity. Other benefits of benralizumab are fast (within 24 h) depletion of peripheral blood eosinophils, potent suppressive activity of bone marrow eosinophils and eosinophil precursors, tissue eosinophil apoptosis regardless of the presence of eosinophil survival factors and even at low IL-5R densities. The fact that benralizumab is dosed subcutaneously and is equally effective when given every eight weeks instead than every four weeks provides patients with convenience of self-administration and make it appealing for patients who dislike injections.

5- Fluorouracil regulation of myeloid-derived suppressor cell differentiation in vitro and in vivo

Abstract The chemotherapeutic agent 5-fluorouracil (5-FU) is the standard therapy for patients with advanced colorectal cancer (CRC). 5-FU not only targets tumor cells for apoptosis but also induces apoptosis in myeloid cells, leading to myelosuppression, which has long been thought as a side effect of 5-FU therapy. Myeloid-derived suppressive cells (MDSCs) are a heterogeneous population of immature myeloid cells that exhibit potent suppressive activity to inhibit T and NK cell function. Recent studies have found that 5-FU suppresses MDSCs in mouse models. However, we observed that MDSCs still massively accumulate in human CRC patients after multiple rounds of 5-FU therapy. We hypothesize that a subset of the heterogeneous MDSCs is resistant to 5-FU and that 5-FU therapy selectively eliminates the sensitive MDSCs, enriching the 5-FU-resistant MDSCs. To test this hypothesis, we made use of both in vitro BM-derived MDSC (BM-MDSC) and in vivo mouse tumor models. We observed that 5-FU therapy significantly decreases CD11b+Gr1+ MDSC accumulation in an orthotopic colon cancer mouse model. BM-MDSCs can be induced by cytokines, including GM-CSF, G-CSF, M-CSF and IL-6, either alone or in combination. Cytological analysis indicates that various cytokines induce BM-MDSC to differentially acquire the morphological appearance of myeloid cell subsets, including macrophage- and granulocyte-like cells. Interestingly, all of these BM-derived MDSCs display resistance to 5-FU-induced apoptosis in vitro following exposure to 5-FU at doses as high as 10 μg/mL. Our data indicates that some MDSC subsets display resistance to 5-FU, providing a rationale to explain the enrichment of MDSCs in human CRC patients following successive treatments with 5-FU.

Development of T cells carrying two complementary chimeric antigen receptors against glypican-3 and asialoglycoprotein receptor 1 for the treatment of hepatocellular carcinoma.

Adoptive immunotherapy leveraging chimeric antigen receptor-modified T (CAR-T) cells holds great promise for the treatment of cancer. However, tumor-associated antigens often have low expression levels in normal tissues, which can cause on-target, off-tumor toxicity. Recently, we reported that GPC3-targeted CAR-T cells could eradicate hepatocellular carcinoma (HCC) xenografts in mice. However, it remains unknown whether on-target, off-tumor toxicity can occur. Therefore, we proposed that dual-targeted CAR-T cells co-expressing glypican-3 (GPC3) and asialoglycoprotein receptor 1 (ASGR1) (a liver tissue-specific protein)-targeted CARs featuring CD3ζ and 28BB (containing both CD28 and 4-1BB signaling domains), respectively, may have reduced on-target, off-tumor toxicity. Our results demonstrated that dual-targeted CAR-T cells caused no cytotoxicity to ASGR1+GPC3- tumor cells, but they exhibited a similar cytotoxicity against GPC3+ASGR1- and GPC3+ASGR1+ HCC cells in vitro. We found that dual-targeted CAR-T cells showed significantly higher cytokine secretion, proliferation and antiapoptosis ability against tumor cells bearing both antigens than single-targeted CAR-T cells in vitro. Furthermore, the dual-targeted CAR-T cells displayed potent growth suppression activity on GPC3+ASGR1+ HCC tumor xenografts, while no obvious growth suppression was seen with single or double antigen-negative tumor xenografts. Additionally, the dual-targeted T cells exerted superior anticancer activity and persistence against single-targeted T cells in two GPC3+ASGR1+ HCC xenograft models. Together, T cells carrying two complementary CARs against GPC3 and ASGR1 may reduce the risk of on-target, off-tumor toxicity while maintaining relatively potent antitumor activities on GPC3+ASGR1+ HCC.

DNA Methyltransferase Inhibitor Promotes Human CD4+CD25hFOXP3+ Regulatory T Lymphocyte Induction under Suboptimal TCR Stimulation.

The “master transcription factor” FOXP3 regulates the differentiation, homeostasis, and suppressor function of CD4+ regulatory T (Treg) cells, which are critical in maintaining immune tolerance. Epigenetic regulation of FOXP3 expression has been demonstrated to be important to Treg cell development, but the induction of human Treg cells through epigenetic modification has not been clearly described. We report that the combination of the DNA methyltransferase inhibitor 5-azacytidine (5-Aza) and suboptimal T cell receptor (TCR) stimulation promoted CD4+CD25hFOXP3+ T cell induction from human CD4+CD25− T cells. 5-Aza treatment enhanced the expression of Treg cell signature genes, such as CD25, FOXP3, CTLA-4, and GITR, in CD4+CD25h cells. Moreover, 5-Aza-treated CD4+CD25h T cells showed potent suppressive activity in a cell contact-dependent manner and reduced methylation in the Treg-specific demethylated region (TSDR) in the FOXP3 gene. The analysis of cytokine production revealed that CD4+CD25− T cells with 5-Aza treatment produced comparable levels of interferon (IFN)-γ and transforming growth factor (TGF)-β, but less IL-10 and more IL-2, when compared to cells without 5-Aza treatment. The increased IL-2 was indispensible to the enhanced FOXP3 expression in 5-Aza-treated CD4+CD25h cells. Finally, 5-Aza-treated CD4+CD25h T cells could be expanded with IL-2 supplementation alone and maintained FOXP3 expression and suppressor function through the expansion. Our findings demonstrate that DNA demethylation can enhance the induction of human Treg cells and promise to solve one of the challenges with using Treg cells in therapeutic approaches.

Myeloid derived suppressor cell: A new player in periodontal disease?

Periodontal disease can be initiated by a shift from a symbiotic to a dysbiotic microbial community. An increase in the recruitment of leukocytes and production of inflammatory cytokines, chemokines and oxidative stress are generated by this shift. In periodontitis, an exacerbated, poorly specific and effective inflammatory response is mounted. Moreover, failure in the inflammation resolving mechanism leads to establishment of a chronic inflammatory process, resulting in the progressive destruction of bone and soft tissue. In different diseases presenting chronic inflammation some important players of immune response are defectives. Thus, an immunosuppressive environment could be induced during chronic inflammation. Myeloid derived suppressor cells (MDSC), a heterogenic group of immature myeloid cells with potent immune suppressive activity, are increased in several acute and chronic inflammatory diseases. Dysbiosis-mediated inflammation can induce increased frequency of MDSC. In addition, mediators generated in diverse inflammatory diseases have demonstrated to promote expansion, activation and recruitment of MDSC, similar mediators have been described in periodontal disease. MDSC promote generation of nitric oxide (NO) and reactive oxygen species (ROS). Furthermore, MDSC can differentiate in functional osteoclasts. We hypothesize that MDSC are generated during periodontal disease. Review of literature evaluating this hypothesis and possible implications are assed in this work. It encourages the study of MDSC in this common disease.

202. Development of T-Cells Carrying Two Complementary Chimeric Antigen Receptors Against GPC3 and ASGR1 for the Treatment of Hepatocellular Carcinoma

Recently, we reported that GPC3-targeted CAR-T cells could eradicate hepatocellular carcinoma xenografts in mice. However, we do not know whether on-target off-tumor toxicity, a major toxicity associated with CAR-T cells will happen when using these GPC3-CAR T cells in human being. We propose that dual-targeted CAR-T cells co-expressing a GPC3- and a ASGR1 (a liver tissue-specific protein)-targeted CAR that signal using CD3ζ and 28BB (containing both CD28 and 4-1BB signaling domain) respectively might be used to reduce on-target off-tumor toxicity. We first demonstrated that GPC3 and ASGR1 were co-expressed in 54.7% of the tested HCC samples. We further demonstrated that dual-targeted CAR-T cells could exhibit no cytotoxic activities on ASGR1+GPC3− tumor cells. However, the cytotoxic activities of GPC3-targeted CAR-T cells and dual-targeted CAR-T cells have similar anti-tumor activities in vitro on ASGR1-GPC3+ or GPC3+ASGR1+HCC cells. Interestingly, dual-targeted CAR-T cells showed significantly higher cytokine secretion, proliferation and anti-apoptosis ability against tumor cells bearing both antigens in vitro than single-targeted CAR-T cells. Furthermore the dual-targeted CAR-T cells displayed potent growth suppression activity on GPC3+ASGR1+HCC tumor xenograft while no obvious growth suppression on single or double antigen-negative tumor xenografts. Additionally, the dual targeting T cells exerted superior anti-cancer activity and persistence than single targeting T cells in two GPC3+ASGR1+ HCC xenograft models. Together, T-cells carrying two complementary CARs against GPC3 and ASGR1 might reduce the risk of on-target off-tumor toxicity while remain relative potent anti-tumor activities on GPC3+ASGR1+ HCC

CD45 phosphatase regulates the fate of myeloid cells in tumor microenvironment by inhibiting STAT3 activity

Abstract The abnormal differentiation of myeloid cells is one of the key features of altered immune response in cancer. Myeloid derived suppressor cells (MDSC) and tumor associated macrophages (TAM) are the major components of immune suppressive network. MDSC accumulate in large numbers in peripheral lymphoid organs and inside tumors. However, in contrast to peripheral lymphoid organs such as spleen, inside tumors, monocytic MDSC rapidly differentiate to TAM with potent immune suppressive activity. We have demonstrated that this phenomenon is mediated by dramatic down-regulation of STAT3 activity in MDSC in tumor site. In cancer patients, MDSC in tumor site also had substantially lower level of pSTAT3 than MDSC in peripheral blood. These results were unexpected, since high level of STAT3 activity is a hallmark of MDSC present in blood and lymphoid organs. The mice with constitutively active STAT3 had shown the expansion of MDSC and down-regulation of macrophage population in tumor microenvironment, emphasizing the crucial role of STAT3 in myeloid cell differentiation. Hypoxia is an intricate part of tumor microenvironment. The decreased STAT3 activity was caused by hypoxia and affected all myeloid cells but was not observed in tumor cells. We further demonstrate that the up-regulation of CD45 tyrosine phosphatase activity in MDSC exposed to hypoxia in tumor site was responsible for down-regulation of STAT3 activity. Moreover, the positive regulation of CD45 activity in hypoxia was mediated by the disruption of CD45 protein dimerization via sialylation. This suggests a novel role of STAT3 in the regulation of myeloid cells in tumors with potential implications for therapeutic targeting.

Targeting T cell senescence in the tumor microenvironment for tumor immunotherapy

Abstract Understanding the molecular mechanisms involved in creating and sustaining the tumor suppressive microenvironment is critical for the development of novel anti-tumor therapeutic strategies. Regulatory T cells (Treg) are a key player for the tumor immunosuppressive microenvironment. However, the molecular processes and functions of suppressed responder T cells mediated by Treg cells remain largely unknown. We discovered a novel suppressive mechanism whereby human Treg cells induce senescence in naïve and effector T cells that then exhibit potent suppressive activity and amplify immune suppression. Treg-induced senescent T cells were distinctly different from the exhausted or anergic T cells based on the transcriptome analyses and phenotypic profiles. We further identified that the nuclear kinase ataxia-telangiectasia mutated protein (ATM)-induced DNA damage was critical and the main cause for the induction of responder T cell senescence and dysfunction mediated by human Treg cells. In addition, MAPK ERK1/2 and p38 were involved in the regulation of senescence process in responder T cells. Importantly, we further revealed that human Treg-induced senescence and suppressor function could be blocked by specific ATM signaling and/or ERK1/2 and p38 signaling inhibition in vitro and in vivo in animal models. Our studies identify a novel molecular mechanism responsible for human Treg cell suppression, and provide new insights relevant for the development of strategies capable of preventing and/or reversing Treg-induced immune suppression for tumor immunotherapy. Funding Support: Supported by the grants from the Melanoma Research Alliance, American Cancer Society and the National Institutes of Health.

Anticancer efficacy of the hypoxia-activated prodrug evofosfamide (TH-302) in osteolytic breast cancer murine models.

Tumor hypoxia is a major cause of treatment failure for a variety of malignancies. However, hypoxia offers treatment opportunities, exemplified by the development of compounds that target hypoxic regions within tumors. Evofosfamide (TH‐302) is a prodrug created by the conjugation of 2‐nitroimidazole to bromo‐isophosphoramide mustard (Br‐IPM). When evofosfamide is delivered to hypoxic regions, the DNA cross‐linking effector, Br‐IPM, is released. This study assessed the cytotoxic activity of evofosfamide in vitro and its antitumor activity against osteolytic breast cancer either alone or in combination with paclitaxel in vivo. A panel of human breast cancer cell lines were treated with evofosfamide under hypoxia and assessed for cell viability. Osteolytic MDA‐MB‐231‐TXSA cells were transplanted into the mammary fat pad, or into tibiae of mice, allowed to establish and treated with evofosfamide, paclitaxel, or both. Tumor burden was monitored using bioluminescence, and cancer‐induced bone destruction was measured using micro‐CT. In vitro, evofosfamide was selectively cytotoxic under hypoxic conditions. In vivo evofosfamide was tumor suppressive as a single agent and cooperated with paclitaxel to reduce mammary tumor growth. Breast cancer cells transplanted into the tibiae of mice developed osteolytic lesions. In contrast, treatment with evofosfamide or paclitaxel resulted in a significant delay in tumor growth and an overall reduction in tumor burden in bone, whereas combined treatment resulted in a significantly greater reduction in tumor burden in the tibia of mice. Evofosfamide cooperates with paclitaxel and exhibits potent tumor suppressive activity against breast cancer growth in the mammary gland and in bone.

Nonclassical CD4+CD49b+ Regulatory T Cells as a Better Alternative to Conventional CD4+CD25+ T Cells To Dampen Arthritis Severity.

Promising immunotherapeutic strategies are emerging to restore tolerance in autoimmune diseases by triggering an increase in the number and/or the function of endogenous regulatory T (Treg) cells, which actively control pathological immune responses. Evidence suggests a remarkable heterogeneity in peripheral Treg cells that warrants their better characterization in terms of phenotype and suppressive function, to determine which subset may be optimally suitable for a given clinical situation. We found that repetitive injections of immature dendritic cells expanded Foxp3-negative CD49b(+) Treg cells that displayed an effector memory phenotype. These expanded Treg cells were isolated ex vivo for transcriptome analysis and found to contain multiple transcripts of the canonical Treg signature shared mainly by CD25(+) but also by other subphenotypes. We characterized the CD49b(+) Treg cell phenotype, underscoring its similarities with the CD25(+) Treg cell phenotype and highlighting some differential expression patterns for several markers, including lymphocyte activation gene 3, KLRG1, CD103, ICOS, CTLA-4, and granzyme B. Comparison of the CD25(+) and CD49b(+) Treg cells' suppressive mechanisms, in vitro and in vivo, revealed the latter's potent suppressive activity, which was partly dependent on IL-10 secretion. Altogether, our results strongly suggest that expression of several canonical Treg cell markers and suppressive function could be Foxp3 independent, and underscore the therapeutic potential of IL-10-secreting CD49b(+) Treg cells in arthritis.

The Synergistic Local Immunosuppressive Effects of Neural Stem Cells Expressing Indoleamine 2,3-Dioxygenase (IDO) in an Experimental Autoimmune Encephalomyelitis (EAE) Animal Model.

Neurodegenerative diseases provoke robust immunological reactions in the central nervous system (CNS), which further deteriorate the neural tissue damage. We hypothesized that the expression levels of indoleamine 2,3-dioxygenase (IDO), an enzyme that has potent immune suppressive activities, in neural stem cells (NSCs) would have synergistic therapeutic effects against neurodegenerative diseases, since NSCs themselves have low IDO expression. In this study, the synergistic immune suppressive effects of rat fetal NSCs expressing IDO (rfNSCs-IDO) were validated by mixed leukocyte reaction (MLR) in vitro and an experimental autoimmune encephalomyelitis (EAE) animal model in vivo. rfNSCs-IDO showed significantly more suppressive effects on T cell proliferation in the MLR compared to control rfNSCs (rfNSCs-Cont). Importantly, IDO inhibition using 1-methyl-DL-tryptophan (1-MT), an IDO inhibitor, reversed the synergistic effects, confirming IDO-specific effects in rfNSCs-IDO. In the EAE animal model, systemic rfNSCs-IDO injections resulted in significant local immune suppression in the cervical lymph nodes and CNS, evidenced by a reduction in the number of activated T lymphocytes and an increase in regulatory T cell numbers, which induced significantly fewer clinical symptoms and faster recovery. In contrast, rfNSCs-Cont failed to reduce symptoms in the EAE animal models, although they showed local immune suppression, which was significantly less than that in rfNSCs-IDO. Taken together, IDO expression in NSCs synergistically potentiates the immune suppression activities of NSCs and could be applicable for the development of therapeutic modalities against various neurodegenerative diseases.

Role for Megakaryocyte Phosphatidylinositol Transfer Proteins-Alpha and -Beta in TGF Beta-Mediated Regulation of Hematopoietic Homeostasis

There are still unknowns regarding homeostatic regulation of hematopoietic stem (HSC) and progenitor (HPC) cells. Deciphering these processes are important for understanding and treating hematopoietic diseases. Phosphatidylinositol is a rare membrane structure lipid, but is critical for cellular signaling upon phosphorylation by lipid kinases to generate phosphoinositide. While phosphoinositide pathways contribute to events linked to the cytoskeleton, little is known of these pathways in regulating hematopoiesis. Critical to this pathway are phosphatidylinositol transfer proteins (PITPs) that in vitro enhance transfer of aqueous insoluble phosphatidylinositol from one membrane to another. Class I PITP proteins PITP α and β are highly conserved, small, and ubiquitously expressed in mammalian cells. To test the hypothesis that phosphatidylinositol signaling contributes to hematopoiesis, we generated conditional knock out mice that lack either PITPα single isoform (PITPαfl/fl PF4Cre+) or both PITPα and PITPβ (PITPαfl/fl βfl/fl PF4Cre+) specifically in their platelets and megakaryocytes, and observed a bone marrow (BM) HSC/HPC phenotype. BM from these mice and their littermate controls were evaluated for absolute numbers of nucleated cells, HSC, and HPC. Cells were analyzed by rigorous phenotyping for long-term (LT)-HSC, short-term (ST)-HSC, multipotential (MPP), common myeloid (CMP), and granulocyte macrophage (GMP) progenitors. They were also assessed for functional HPC by colony assays in vitro for multi-cytokine (Epo, GM-CSF, IL-3, SCF, hemin) stimulated granulocyte macrophage (CFU-GM), erythroid (BFU-E), and multipotential (CFU-GEMM) progenitors, and for their cycling status using a high specific activity tritiated thymidine kill assay. PITPα-/-, and to a greater extent PITPα/β-/-, progenitor cells demonstrated significant decreases in LT-HSC and ST-HSC per femur. While there were no significant changes in numbers of MPP, CMP, and GMP in the PITPα and PITPα/β-/- BM compared to controls, there were significant decreases of approximately 50% in numbers of CFU-GM, BFU-E, and CFU-GEMM per femur. PITC-/- HPC were in a slow or non-cycling state compared to the rapid cell cycle (40-57% in S-phase) of control HPC. Thus PITPα-/- and PITPα/β -/- BM cells were associated with decreased HSC and functional HPC numbers. To evaluate mechanisms for this phenotype, we focused on BM megakaryocytes, as they have been implicated in microenvironmental regulation of hematopoiesis, and PITPα and PITPα/β activities are associated with megakaryocyte/platelet function. BM derived TPO-culture expanded megakaryocytes were allowed to condition medium for 48 hours, and conditioned medium (CM) from PITPα-/-, PITPα/β-/-, and control BM megakaryocytes were assayed for effects on colony formation by multicytokine stimulated BM cells derived from normal mice. CM from PITPα-/- and PITPα/β-/- megakaryocytes, but not from control mice, significantly suppressed colony formation by CFU-GM, BFU-E and CFU-GEMM (by ~50%). Limiting dilution analysis of the CM demonstrated that PITPα/β-/- cells had more potent suppressor activity than PITPα-/- cells. Bioplex analysis of the CM from PITPα -/- and PITPα/β -/- megakaryocytes demonstrated significantly higher levels of cytokines/chemokines with known myelosuppressive activities (including: TNF-α, VEGF, LIF, IP-10, ENA-78, MDC, MIG, and MIP-1α). However, ELISA analysis of TGF-β1, demonstrated minimal protein in BM flushes from control mice, but large amounts of TGF-β (>350 pg/ml) in BM flushes from the PITPα/β -/- mice. CM from PITPα and α/β-/- megakaryocytes also contained highly elevated TGF-β protein. Thus, we hypothesized that the effect of PITP -/- on the suppression of HPC colony formation was mediated by TGF-β. The myelosuppressive CM derived from PITPα and PITPα/β -/- megakaryocytes was completely neutralized by a monoclonal TGF-β antibody. This demonstrates that PITPα and PITP α/β-/- megakaryocytes produce elevated TGF-β that at least in part, and possibly in synergy with other myelosuppressive cytokines/chemokines, decreases numbers of HSC and functional HPC. Our studies demonstrate a link between PITPα and α/β and TGF-β levels with significant effects on HSCs and HPCs, thus demonstrating involvement of the phosphoinositide pathway in homeostatic regulation of hematopoiesis. DisclosuresNo relevant conflicts of interest to declare.

Tolerance and immune suppression in the tumor microenvironment

The concept of immunological tolerance has guided and permeated much of modern immunology. Ray Owen’s ground-breaking observations in twin cattle provided the first mechanistic explanation for tolerance to self-molecules and established tolerance as a beneficial process that protects the host against autoreactivity. However, his studies also opened the door to understanding that tolerance may be detrimental, such as occurs when cancer cells induce tolerance/immune suppression resulting in inhibition of anti-tumor immunity. This article briefly traces the early history of the field of tumor immunology with respect to tolerance, and then focuses on a relatively recently identified population of cells called myeloid-derived suppressor cells (MDSCs). MDSC are instrumental in causing tolerance/immune suppression in individuals with cancer. They are present in most individuals with cancer and because of their potent immune suppressive activity are a major deterrent to natural anti-tumor immunity and a significant obstacle to immunotherapy.

Myeloid-derived suppressor cells contribute to bone erosion in collagen-induced arthritis by differentiating to osteoclasts

Bone erosion is a sign of severe rheumatoid arthritis and osteoclasts play a major role in the bone resorption. Recently, myeloid-derived suppressor cells (MDSC) has been reported to be increased in collagen-induced arthritis (CIA). The number of circulating MDSCs is shown to correlate with rheumatoid arthritis. These findings suggest that MDSCs are precursor cells involved in bone erosion. In this study, MDSCs isolated from mice with CIA stimulated with M-CSF and RANKL in vitro expressed osteoclast markers and acquired osteoclast bone resorption function. MDSCs sorted from CIA mice were transferred into the tibia of normal DBA/1J mice and bones were subjected to histological and Micro CT analyses. The transferred CIA-MDSCs were shown to differentiate into TRAP+ osteoclasts that were capable of bone resorption in vivo. MDSCs isolated from normal mice had more potent suppressor activity and much less capability to differentiate to osteoclast. Additional experiments showed that NF-κB inhibitor Bay 11–7082 or IκB inhibitor peptide blocked the differentiation of MDSCs to osteoclast and bone resorption. IL-1Ra also blocked this differentiation. In contrast, the addition of IL-1α further enhanced osteoclast differentiation and bone resorption. These results suggest that MDSCs are a source of osteoclast precursors and inflammatory cytokines such as IL-1, contributing significantly to erosive changes seen in rheumatoid arthritis and related disorders.

Abstract 2343: Tumor microenvironment regulates the fate of myeloid cells by controlling STAT3 activity

Abstract The abnormal differentiation of myeloid cells is one of the key features of altered immune response in cancer. Myeloid derived suppressor cells (MDSC) are a major component of immune suppressive network. MDSC accumulate in large numbers in peripheral lymphoid organs and inside tumors. Previous data indicated that the fate of MDSC depends on the localization of these cells. In contrast to peripheral lymphoid organs such as spleen, inside tumors, MDSC rapidly differentiate to tumor associated macrophages with potent immune suppressive activity. We have demonstrated that this phenomenon is mediated by dramatic down-regulation of STAT3 activity in MDSC at tumor site. In cancer patients, MDSC at tumor site also had substantially lower level of pSTAT3 than MDSC in peripheral blood. These results were unexpected, since high level of STAT3 activity is a hallmark of MDSC present in blood and lymphoid organs. The mice with constitutively active STAT3 had shown the dramatic expansion of MDSC and down-regulation of macrophage population in tumor microenvironment, emphasizing the crucial role of STAT3 in myeloid cell differentiation. Hypoxia is an intricate part of tumor microenvironment. We have found that hypoxia caused the down-regulation of STAT3 activity in MDSC. However, the effect of hypoxia on STAT3 activity was not observed in tumor cells. We further demonstrate that the up-regulation of CD45 tyrosine phosphatase activity in MDSC exposed to hypoxia in tumor site was responsible for down-regulation of STAT3 activity. Using KO model we have shown that MDSC devoid of CD45 failed to experience the effect of hypoxia on pSTAT3 down-regulation as well as their differentiation to macrophage at tumor site. Furthermore, the positive regulation of CD45 activity in hypoxia was mediated by the disruption of CD45 protein dimerization via sialylation. The treatment of sialidase abrogated the effect of hypoxia on STAT3 activity in MDSC and differentiation of MDSC to macrophages. This novel mechanism of regulation of myeloid cell differentiation by STAT3 may provide rationale for re-evaluation of therapeutic strategies in cancer. Citation Format: Vinit Kumar, Pingyan Cheng, Thomas Condamine, Dmitry Gabrilovich. Tumor microenvironment regulates the fate of myeloid cells by controlling STAT3 activity. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2343. doi:10.1158/1538-7445.AM2015-2343

The Role of Dendritic Cells in Central Tolerance.

Dendritic cells (DCs) play a significant role in establishing self-tolerance through their ability to present self-antigens to developing T cells in the thymus. DCs are predominantly localized in the medullary region of thymus and present a broad range of self-antigens, which include tissue-restricted antigens expressed and transferred from medullary thymic epithelial cells, circulating antigens directly captured by thymic DCs through coticomedullary junction blood vessels, and peripheral tissue antigens captured and transported by peripheral tissue DCs homing to the thymus. When antigen-presenting DCs make a high affinity interaction with antigen-specific thymocytes, this interaction drives the interacting thymocytes to death, a process often referred to as negative selection, which fundamentally blocks the self-reactive thymocytes from differentiating into mature T cells. Alternatively, the interacting thymocytes differentiate into the regulatory T (Treg) cells, a distinct T cell subset with potent immune suppressive activities. The specific mechanisms by which thymic DCs differentiate Treg cells have been proposed by several laboratories. Here, we review the literatures that elucidate the contribution of thymic DCs to negative selection and Treg cell differentiation, and discusses its potential mechanisms and future directions.