Johanna K. Morales

GM-CSF is one of the main breast tumor-derived soluble factors involved in the differentiation of CD11b-Gr1- bone marrow progenitor cells into myeloid-derived suppressor cells

Recent reports have shown the involvement of tumor burden as well as GM-CSF in supporting myeloid-derived suppressor cells (MDSC). However, it is not known what progenitor cells may differentiate into MDSC in the presence of GM-CSF, and whether FVBN202 transgenic mouse model of spontaneous breast carcinoma may exhibit distinct subset distribution of CD11b+Gr1+ cells. In addition, it is not known why CD11b+Gr1+ cells derived from tumor-free and tumor-bearing animals exhibit different functions. In this study, we determined that GM-CSF was one of the tumor-derived soluble factors that induced differentiation of CD11b-Gr1- progenitor cells from within monocytic/granulocytic bone marrow cells into CD11b+Gr1+ cells. We also showed that CD11b+Gr1+ cells in FVBN202 mice consisted of CD11b+Ly6G-Ly6C+ suppressive and CD11b+Ly6G+Ly6C+ non-suppressive subsets. Previously reported variations between tumor-free and tumor-bearing animals in the function of their CD11b+Gr1+ cells were found to be due to the variations in the proportion of these two subsets. Therefore, increasing ratios of CD11b+Gr1+ cells derived from tumor-free animals revealed their suppressive activity on T cells, in vitro. Importantly, GM-CSF supported the generation of CD11b+Ly6G-Ly6C+ suppressor subsets that inhibited proliferation as well as anti-tumor function of neu-specific T cells. These findings suggest revisiting the use of GM-CSF for the expansion of dendritic cells, ex vivo, for cell-based immunotherapy or as an adjuvant for vaccines for patients with cancer in whom MDSC play a major role in the suppression of anti-tumor immune responses.

Essential roles of sphingosine-1–phosphate receptor 2 in human mast cell activation, anaphylaxis, and pulmonary edema

Allergic disease is endemic in developed nations, and none is more dramatic or deadly than systemic anaphylactic shock (Finkelman, 2007). Unfortunately, this collection of rapid changes to the cutaneous, vasculature, and pulmonary systems may also be the least understood of atopic diseases. A better understanding of the eliciting factors for systemic anaphylaxis is therefore critical for accurate diagnosis and therapeutic intervention. Mast cells, which reside in vascularized tissues and are strategically located at the interfaces of host and environment in skin and mucosal surfaces, are key effectors of IgEmediated allergic disorders, including anaphylaxis, hay fever, eczema, and asthma. Mast cells express FcRI (high-affinity receptors for IgE), which binds antigen (Ag)-specific IgE antibodies, and subsequent exposure to Ag initiates cross-linking and aggregation of FcRI. This cascade of events triggers mast cell activation and secretion of a wide array of inflammatory mediators, such as histamine and other preformed mediators, bioactive lipids, including eicosanoids and platelet-activating factor (PAF), and numerous proinflammatory cytokines and chemokines (Kalesnikoff and Galli, 2008), which are essential to the pathogenesis of allergic diseases and anaphylaxis (Finkelman, 2007). A recent addition to the recognized repertoire of lipid mediators secreted by mast cells is the sphingolipid metabolite sphingosine1–phosphate (S1P; Jolly et al., 2004; Mitra et al., 2006; Olivera et al., 2006), which has been implicated in initiation and maintenance of diverse aspects of immune cell activation and function (Rosen et al., 2008; for review see Schwab and Cyster, 2007; Rivera et al., 2008). Focus on S1P in immune responses has increased after the elucidation of its critical role in lymphocyte trafficking (Rosen et al., 2008; for review see Schwab and Cyster, 2007), and observations of local increases of S1P in inflammatory disorders such as asthma (Ammit et al., 2001) and rheumatoid arthritis (Kitano et al., 2006). S1P is a ligand for five G protein–coupled receptors, designated S1P1-5, through which it exerts many of its actions (Spiegel and Milstien, 2003). CORRESPONDENCE Carole Oskeritzian: coskerit@vcu.edu

Cutting Edge: Mast Cells Critically Augment Myeloid-Derived Suppressor Cell Activity

Myeloid-derived suppressor cells (MDSCs) are primarily recognized for their immunosuppressive properties in malignant disease. However, their interaction with other innate immune cells and their regulation of immune responses, such as in parasitic infection, necessitate further characterization. We used our previously published mouse model of MDSC accumulation to examine the immunoregulatory role of MDSCs in B16 melanoma metastasis and Nippostrongylus brasiliensis infection. In this study, we demonstrate that the activity of MDSCs is dependent on the immune stimuli and subset induced. Monocytic MDSCs predictably suppressed antitumor immune responses but granulocytic MDSCs surprisingly enhanced the clearance of N. brasiliensis infection. Intriguingly, both results were dependent on MDSC interaction with mast cells (MCs), as demonstrated by adoptive-transfer studies in MC-deficient (KitWsh/Wsh) mice. These findings were further supported by ex vivo cocultures of MCs and MDSCs, indicating a synergistic increase in cytokine production. Thus, MCs can enhance both immunosuppressive and immunosupportive functions of MDSCs.

Adoptive transfer of HER2/neu-specific T cells expanded with alternating gamma chain cytokines mediate tumor regression when combined with the depletion of myeloid-derived suppressor cells

Adoptive immunotherapy (AIT) using ex vivo-expanded HER-2/neu-specific T cells has shown initial promising results against disseminated tumor cells in the bone marrow. However, it has failed to promote objective responses against primary tumors. We report for the first time that alternating gamma chain cytokines (IL-2, IL-7 and IL-15) ex vivo can expand the neu-specific lymphocytes that can kill breast tumors in vitro. However, the anti-tumor efficacy of these neu-specific T cells was compromised by the increased levels of myeloid-derived suppressor cells (MDSC) during the premalignant stage in FVBN202 transgenic mouse model of breast carcinoma. Combination of AIT with the depletion of MDSC, in vivo, resulted in the regression of neu positive primary tumors. Importantly, neu-specific antibody responses were restored only when AIT was combined with the depletion of MDSC. In vitro studies determined that MDSC caused inhibition of T cell proliferation in a contact-dependent manner. Together, these results suggest that combination of AIT with depletion or inhibition of MDSC could lead to the regression of mammary tumors.

IL-4 and TGF-β1 Counterbalance One Another while Regulating Mast Cell Homeostasis

Mast cell responses can be altered by cytokines, including those secreted by Th2 and regulatory T cells (Treg). Given the important role of mast cells in Th2-mediated inflammation and recent demonstrations of Treg-mast cell interactions, we examined the ability of IL-4 and TGF-β1 to regulate mast cell homeostasis. Using in vitro and in vivo studies of mouse and human mast cells, we demonstrate that IL-4 suppresses TGF-β1 receptor expression and signaling, and vice versa. In vitro studies demonstrated that IL-4 and TGF-β1 had balancing effects on mast cell survival, migration, and FcεRI expression, with each cytokine cancelling the effects of the other. However, in vivo analysis of peritoneal inflammation during Nippostrongylus brasiliensis infection in mice revealed a dominant suppressive function for TGF-β1. These data support the existence of a cytokine network involving the Th2 cytokine IL-4 and the Treg cytokine TGF-β1 that can regulate mast cell homeostasis. Dysregulation of this balance may impact allergic disease and be amenable to targeted therapy.

Danger signals and nonself entity of tumor antigen are both required for eliciting effective immune responses against HER-2/neu positive mammary carcinoma: implications for vaccine design.

Using parental FVB mice and their neu transgenic counterparts, FVBN202, we showed for the first time that dangerous hyperplasia of mammary epithelial cells coincided with breaking immunological tolerance to the neu “self” tumor antigen, though such immune responses failed to prevent formation of spontaneous neu-overexpressing mammary carcinoma (MMC) or reject transplanted MMC in FVBN202 mice. On the other hand, neu-specific immune responses appeared to be effective against MMC in parental FVB mice because of the fact that rat neu protein was seen as “nonself” antigen in these animals and the protein was dangerously overexpressed in MMC. Interestingly, low/intermediate expression of the neu “nonself” protein in tumors induced immune responses but such immune responses failed to reject the tumor in FVB mice. Our results showed that self–nonself (SNS) entity of a tumor antigen or danger signal alone, while may equally induce an antigen-specific immune response, will not warrant the efficacy of immune responses against tumors. On the other hand, entity of antigen in the context of dangerous conditions, i.e. abnormal/dangerous overexpression of the neu nonself protein, will warrant effective anti-tumor immune responses in FVB mice. This unified “danger-SNS” model suggests focusing on identification of naturally processed cryptic or mutated epitopes, which are considered semi-nonself by the host immune system, along with novel dangerous adjuvant in vaccine design.

The Fyn-STAT5 pathway: a new Frontier in IgE- and IgG-mediated mast cell signaling

Mast cells are central players in immune surveillance and activation, positioned at the host–environment interface. Understanding the signaling events controlling mast cell function, especially those that maintain host homeostasis, is an important and still less understood area of mast cell-mediated disease. With respect to allergic disease, it is well established that IgE and its high affinity receptor FcεRI are major mediators of mast cell activation. However, IgG-mediated signals can also modulate mast cell activities. Signals elicited by IgG binding to its cognate receptors (FcγR) are the basis for autoimmune disorders such as lupus and rheumatoid arthritis. Using knowledge of IgE-mediated mast cell signaling, recent work has begun to illuminate potential overlap between FcεRI and FcγR signal transduction. Herein we review the importance of Src family kinases in FcεRI and FcγR signaling, the role of the transcription factor STAT5, and impingement of the regulatory cytokines IL-4, IL-10, and TGFβ1 upon this network.

Myeloid-derived suppressor cells enhance IgE-mediated mast cell responses

Mast cells and MDSCs are increased by parasitic infection and tumor growth. We previously demonstrated that enhanced MDSC development in ADAM10 transgenic mice yielded resistance to Nb infection and that coculturing MDSCs and mast cells enhanced cytokine production. In the current work, we show that MDSC‐mast cell coculture selectively enhances IgE‐mediated cytokine secretion among mast cells, without increasing MDSC cytokine production. This effect was independent of cell contact and elicited by Ly6C+ and Ly6C/G+ MDSC subsets. These interactions were functionally important. MDSC depletion with the FDA‐approved drug gemcitabine exacerbated Nb or Trichinella spiralis infection and reduced mast cell‐dependent AHR and lung inflammation. Adoptive transfer of MDSC worsened AHR in WT but not mast cell‐deficient Wsh/Wsh mice. These data support the hypothesis that MDSCs enhance mast cell inflammatory responses and demonstrate that this interaction can be altered by an existing chemotherapeutic.

Mast Cell Regulation of the Immune Response

Mast cells are well known as principle effector cells of type I hypersensitivity responses. Beyond this role in allergic disease, these cells are now appreciated as playing an important role in many inflammatory conditions. This review summarizes the support for mast cell involvement in resisting bacterial infection, exacerbating autoimmunity and atherosclerosis, and promoting cancer progression. A commonality in these conditions is the ability of mast cells to elicit migration of many cell types, often through the production of inflammatory cytokines such as tumor necrosis factor. However, recent data also demonstrates that mast cells can suppress the immune response through interleukin-10 production. The data encourage those working in this field to expand their view of how mast cells contribute to immune homeostasis.

IgE signaling suppresses FcεRIβ expression

Activation of the high‐affinity receptor for IgE, FcεRI, is known to elicit its rapid down‐regulation through internalization and degradation. In keeping with this, expression of all three FcεRI subunits is decreased at the protein level after cross‐linkage of IgE with antigen. However, we find that the FcεRI β‐subunit is also selectively suppressed at the mRNA level, through a pathway primarily involving Fyn, Syk, PI3K, and NF‐κB. IgG or calcium ionophore, stimuli known to mimic portions of the IgE signaling cascade, similarly suppressed β‐subunit expression. LPS, a NF‐κB‐activating TLR ligand, did not alter β‐subunit expression. As IgE increases FcεRI expression, we examined the coordinated regulation of FcεRI subunits during culture with IgE, followed by cross‐linkage with antigen. IgE increased the expression of all three FcεRI subunits and strikingly induced expression of the antagonistic βT. The ratio of β:βT protein expression decreased significantly during culture with IgE and was reset to starting levels by antigen cross‐linkage. These changes in protein levels were matched by similar fluctuations in β and βT mRNAs. FcεRIβ is a key regulator of IgER expression and function, a gene in which polymorphisms correlate with allergic disease prevalence. The ability of IgE and FcεRI signaling to coordinate expression of the β and βT subunits may comprise a homeostatic feedback loop—one that could promote chronic inflammation and allergic disease if dysregulated.