Carlo Pucillo

Mast Cells Boost Myeloid-Derived Suppressor Cell Activity and Contribute to the Development of Tumor-Favoring Microenvironment

Danelli and colleagues demonstrate interactions between mast cells and myeloid-derived suppressor cells in the mucosa of colon carcinoma patients and in the colon and spleen of tumor-bearing mice and establish the role of CD40/CD40L in the activity of these cells in colon cancer. Inflammation plays crucial roles at different stages of tumor development and may lead to the failure of immune surveillance and immunotherapy. Myeloid-derived suppressor cells (MDSC) are one of the major components of the immune-suppressive network that favors tumor growth, and their interaction with mast cells is emerging as critical for the outcome of the tumor-associated immune response. Herein, we showed the occurrence of cell-to-cell interactions between MDSCs and mast cells in the mucosa of patients with colon carcinoma and in the colon and spleen of tumor-bearing mice. Furthermore, we demonstrated that the CT-26 colon cancer cells induced the accumulation of CD11b+Gr1+ immature MDSCs and the recruitment of protumoral mast cells at the tumor site. Using ex vivo analyses, we showed that mast cells have the ability to increase the suppressive properties of spleen-derived monocytic MDSCs, through a mechanism involving IFNγ and nitric oxide production. In addition, we demonstrated that the CD40:CD40L cross-talk between the two cell populations is responsible for the instauration of a proinflammatory microenvironment and for the increase in the production of mediators that can further support MDSC mobilization and tumor growth. In light of these results, interfering with the MDSC:mast cell axis could be a promising approach to abrogate MDSC-related immune suppression and to improve the antitumor immune response. Cancer Immunol Res; 3(1); 85–95. ©2014 AACR.

CD4+CD25+ regulatory T cells suppress mast cell degranulation and allergic responses through OX40-OX40L interaction

T regulatory (Treg) cells play a role in the suppression of immune responses, thus serving to induce tolerance and control autoimmunity. Here, we explored whether Treg cells influence the immediate hypersensitivity response of mast cells (MCs). Treg cells directly inhibited the FcvarepsilonRI-dependent MC degranulation through cell-cell contact involving OX40-OX40L interactions between Treg cells and MCs, respectively. When activated in the presence of Treg cells, MCs showed increased cyclic adenosine monophosphate (cAMP) concentrations and reduced Ca(2+) influx, independently of phospholipase C (PLC)-gamma2 or Ca(2+) release from intracellular stores. Antagonism of cAMP in MCs reversed the inhibitory effects of Treg cells, restoring normal Ca(2+) responses and degranulation. Importantly, the in vivo depletion or inactivation of Treg cells caused enhancement of the anaphylactic response. The demonstrated crosstalk between Treg cells and MCs defines a previously unrecognized mechanism controlling MC degranulation. Loss of this interaction may contribute to the severity of allergic responses.

Mast cells counteract regulatory T-cell suppression through interleukin-6 and OX40/OX40L axis toward Th17-cell differentiation

The development of inflammatory diseases implies inactivation of regulatory T (Treg) cells through mechanisms that still are largely unknown. Here we showed that mast cells (MCs), an early source of inflammatory mediators, are able to counteract Treg inhibition over effector T cells. To gain insight into the molecules involved in their interplay, we set up an in vitro system in which all 3 cellular components were put in contact. Reversal of Treg suppression required T cell-derived interleukin-6 (IL-6) and the OX40/OX40L axis. In the presence of activated MCs, concomitant abundance of IL-6 and paucity of Th1/Th2 cytokines skewed Tregs and effector T cells into IL-17-producing T cells (Th17). In vivo analysis of lymph nodes hosting T-cell priming in experimental autoimmune encephalomyelitis revealed activated MCs, Tregs, and Th17 cells displaying tight spatial interactions, further supporting the occurrence of an MC-mediated inhibition of Treg suppression in the establishment of Th17-mediated inflammatory responses.

Gamma-delta T-cell lymphomas

Peripheral T-cell lymphomas (TCLs) are uncommon neoplasms, accounting for about 12% of all lymphoid tumors worldwide. TCLs in which γδ T-cell receptors are expressed (γδ TCLs) are extremely aggressive and rare (<1% of lymphoid neoplasms). γδ TCLs originate from γδ T cells, a small subset of peripheral T cells with direct antigen recognition capability acting at the interface between innate and adaptive immunity. Two distinct γδ TCL entities are recognized: hepatosplenic T-cell lymphoma (HSTL) and primary cutaneous γδ T-cell lymphoma (PCGD-TCL). HSTL is a well-characterized extranodal lymphoma that has a disguised onset, secondary to intrasinusoidal infiltration of the spleen, liver and bone marrow, has a rapidly progressive course that is poorly responsive to chemotherapy, and often ensues in the setting of immune system suppression. PCGD-TCL can present with prominent epidermal involvement or with a panniculitis-like clinical picture that can be complicated by a concurrent hemophagocytic syndrome; the disease shows biological and phenotypic overlap with other extranodal γδ TCLs that involve the respiratory or gastrointestinal tract mucosa. The regular application of phenotypic and molecular techniques is crucial for the diagnosis of γδ TCLs. In this Review, we discuss the clinical and biological features, the diagnostic challenges and the therapeutic perspectives of HSTL and PCGD-TCL.

Redox Potential Controls the Structure and DNA Binding Activity of the Paired Domain

Pax proteins are transcriptional regulators controlling a variety of cell fates during animal development. This role depends on the intact function of the paired (Prd) domain that is able to recognize specific DNA sequences. The Prd domain is composed of two distinct helix-turn-helix subdomains, PAI and RED. Molecular functions of Pax proteins are subjected to different levels of regulation involving both pre-translational and post-translational mechanisms. By using Pax-5 and Pax-8 recombinant proteins, we demonstrate that the binding activity of the Prd domain is regulated through the oxidation/reduction of conserved cysteine residues. Mass spectrometry analysis and mutagenesis experiments demonstrate that the redox regulation is accomplished through the reversible formation of an intramolecular disulfide bridge involving the cysteines present in the PAI subdomain, whereas the RED subdomain appears quite insensitive to redox potential. Circular dichroism experiments indicate that only the reduced form of the Prd domain is able to undergo the proper conformational change necessary for sequence-specific DNA binding. Nuclear extracts from different cell lines contain an activity that is able to reduce the Paired domain and, therefore, to control the DNA binding activity of this protein. Immunodepletion of nuclear extracts demonstrate that the protein Ref-1 contributes to the redox regulation of the Prd DNA binding activity. Given the modular nature of the Prd domain and the independent DNA binding specificity of the PAI and RED subdomains, we propose that this control mechanism should be involved in “switching” among different DNA sequences and therefore different target genes.

H2O2 induces translocation of APE/Ref-1 to mitochondria in the Raji B-cell line

Reactive oxygen species (ROS) are generated as by‐products of respiration and are used as signal transducing intermediates in out–in signaling pathways. ROS are also generated during inflammatory responses and it has been shown that hydrogen peroxide may trigger activation of B‐lymphocytes, similar to cross‐linking of surface immunoglobulins. On the other hand, both exogenous and endogenous generated ROS are a major source of nuclear and mitochondrial DNA (mtDNA) damage. The base excision repair (BER) enzyme APE/Ref‐1 normally repairs small nuclear DNA lesion such as oxidized or alkylated bases. It is not clear though whether DNA repair mechanisms able to abolish oxidative damage from nuclear DNA are present into mitochondria too. Here we show by confocal microscopy and Western blot analysis that in the B‐lymphocyte Raji cell line a fraction of APE/Ref‐1 rapidly re‐localizes into mitochondria following H2O2 activation. Targeting of APE/Ref‐1 to mitochondria is not associated with cytochrome‐c loss or apoptosis induction. These findings indicate that the APE/Ref‐1 translocates to mitochondria in response to oxidative stress and thereby it might exert a protective function.

Studies with a monoclonal antibody to the β subunit of the receptor with high affinity for immunoglobulin E

The receptor with high affinity for IgE consists of a tetrameric complex of polypeptides, one of which (alpha), contains the binding site for IgE. The function of the other chains--a single beta and two disulfide-linked gamma chains--is unknown. We report the cloning of a murine hybridoma that secretes an IgG1 antibody which specifically reacts with the beta subunit. Studies with this monoclonal antibody show that the subunit stoichiometry of the receptor is unaffected by the presence or absence of bound IgE. We also found that under certain conditions where the alpha beta gamma 2 complex dissociates, beta remains attached to the dimer of gamma chains, indicating that these chains contact each other in the native receptor. In rat basophilic leukemia cells--a neoplastic line of mucosal-type mast cells--all of the beta subunits expressed by the cells appeared to be associated with the high affinity receptor. However, in at least one cell line which has no high affinity receptors--a putative rat lymphoma line--beta or beta-like polypeptides were also expressed.

Mast cells enhance proliferation of B lymphocytes and drive their differentiation toward IgA-secreting plasma cells

The evidence of a tight spatial interaction between mast cells (MCs) and B lymphocytes in secondary lymphoid organs, along with the data regarding the abundance of MCs in several B-cell lymphoproliferative disorders prompted us to investigate whether MCs could affect the proliferation and differentiation of B cells. To this aim, we performed coculture assays using mouse splenic B cells and bone marrow-derived MCs. Both nonsensitized and activated MCs proved able to induce a significant inhibition of cell death and an increase in proliferation of naive B cells. Such proliferation was further enhanced in activated B cells. This effect relied on cell-cell contact and MC-derived interleukin-6 (IL-6). Activated MCs could regulate CD40 surface expression on unstimulated B cells and the interaction between CD40 with CD40 ligand (CD40L) on MCs, together with MC-derived cytokines, was involved in the differentiation of B cells into CD138(+) plasma cells and in selective immunoglobulin A (IgA) secretion. These data were corroborated by in vivo evidence of infiltrating MCs in close contact with IgA-expressing plasma cells within inflamed tissues. In conclusion, we reported here a novel role for MCs in sustaining B-cell expansion and driving the development of IgA-oriented humoral immune responses.

Selective Activation of Fyn/PI3K and p38 MAPK Regulates IL-4 Production in BMMC under Nontoxic Stress Condition

Mast cells have the ability to react to multiple stimuli, implicating these cells in many immune responses. Specific signals from the microenvironment in which mast cells reside can activate different molecular events that govern distinct mast cells responses. We previously demonstrated that hydrogen peroxide (H2O2) promotes IL-4 and IL-6 mRNA production and potentates FcεRI-induced cytokine release in rat basophilic leukemia RBL-2H3 cells. To further evaluate the effect of an oxidative microenvironment (which is physiologically present in an inflammatory site) on mast cell function and the molecular events responsible for mast cell cytokine production in this environment, we analyzed the effect of H2O2 treatment on IL-4 production in bone marrow-derived, cultured mast cells. Our findings show that nanomolar concentrations of H2O2 induce cytokine secretion and enhance IL-4 production upon FcεRI triggering. Oxidative stimulation activates a distinct signal transduction pathway that induces Fyn/PI3K/Akt activation and the selective phosphorylation of p38 MAP kinase. Moreover, H2O2 induces AP-1 and NFAT complexes that recognize the IL-4 promoter. The absence of Fyn and PI3K or the inhibition of p38 MAPK activity demonstrated that they are essential for H2O2-driven IL-4 production. These findings show that mast cells can respond to an oxidative microenvironment by initiating specific signals capable of eliciting a selective response. The findings also demonstrate the dominance of the Fyn/p38 MAPK pathway in driving IL-4 production.

The Controlling Roles of Trp60 and Trp95 in β2-Microglobulin Function, Folding and Amyloid Aggregation Properties

Amyloidosis associated to hemodialysis is caused by persistently high beta(2)-microglobulin (beta(2)m) serum levels. beta(2)m is an intrinsically amyloidogenic protein whose capacity to assemble into amyloid fibrils in vitro and in vivo is concentration dependent; no beta(2)m genetic variant is known in the human population. We investigated the roles of two evolutionary conserved Trp residues in relation to beta(2)m structure, function and folding/misfolding by means of a combined biophysical and functional approach. We show that Trp60 plays a functional role in promoting the association of beta(2)m in class I major histocompatibility complex; it is exposed to the solvent at the apex of a protein loop in order to accomplish such function. The Trp60-->Gly mutation has a threefold effect: it stabilizes beta(2)m, inhibits beta(2)m amyloidogenic propensity and weakens the interaction with the class I major histocompatibility complex heavy chain. On the contrary, Trp95 is buried in the beta(2)m core; the Trp95-->Gly mutation destabilizes the protein, which is unfolded in solution, yielding nonfibrillar beta(2)m aggregates. Trp60 and Trp95 therefore play differential and complementary roles in beta(2)m, being relevant for function (Trp60) and for maintenance of a properly folded structure (Trp95) while affecting in distinct ways the intrinsic propensity of wild-type beta(2)m towards self-aggregation into amyloid fibrils.