Cristiana Caorsi

Lactoferrin, a major defense protein of innate immunity, is a novel maturation factor for human dendritic cells

Lactoferrin (LF) is an important protein component of the innate immune system that is broadly distributed within the body fluids. LF is endowed with multiple biological activities. Talactoferrin (TLF), a recombinant human LF, is in clinical development as an anticancer agent and is entering Phase III clinical trials. Here, we show that TLF induces the maturation of human dendritic cells (DCs) derived from monocytes. TLF, at physiologically relevant concentrations (100 µg/ml) up‐regulates the expression of human leukocyte antigen (HLA) class II, CD83, CD80, and CD86 costimulatory molecule and CXCR4 and CCR7 chemokine receptors, acting primarily through the p38 MAPK signaling pathway. DCs matured by TLF displayed an enhanced release of IL‐8 and CXCL10, as well as a significantly reduced production of IL‐6, IL‐10, and CCL20. They also display a reduced ability to take up antigen and increased capacity to trigger proliferation and release IFN‐γ in the presence of allogeneic human T cells. TLF‐matured DCs are able to prime naive T cells to respond to KLH antigen and display a significantly increased capacity to present Flu‐MA58–66 peptide to HLA‐A2‐matched T cells. These data suggest that a key immunomodulatory function that may be mediated by TLF is to link the innate with adaptive immunity through DC maturation.—Spadaro, M., Caorsi, C., Ceruti, P., Varadhachary, A., Forni, G., Pericle, F., Giovarelli, M. Lactoferrin, a major defense protein of innate immunity, is a novel maturation factor for human dendritic cells. FASEB J. 22, 2747–2757 (2008)

Human mesenchymal stem cell-derived microvesicles modulate T cell response to islet antigen glutamic acid decarboxylase in patients with type 1 diabetes

Aims/hypothesisMesenchymal stem cells (MSCs) have been shown to abrogate in vitro the proinflammatory response in type 1 diabetes. The mechanism involves paracrine factors, which may include microvesicles (MVs). We evaluated whether MVs derived from heterologous bone-marrow MSCs exert an immunomodulatory effect on T cell responses against GAD (glutamic acid decarboxylase) antigen in type 1 diabetes.MethodsMVs were purified from heterologous human MSCs by differential centrifugation. Peripheral blood mononuclear cells (PBMCs) were obtained from patients with type 1 diabetes at disease onset, and responses to GAD65 stimulation were assessed by IFN-γ enzyme-linked immunosorbent spot analysis. Levels of cytokines and prostaglandin E2 (PGE2) were measured in the supernatant fraction, and T helper 17 (Th17) and regulatory T cell analysis was performed.ResultsMVs were internalised by PBMCs, as assessed by confocal microscopy and flow cytometry analyses. MVs significantly decreased IFN-γ spots and levels in GAD65-stimulated PBMCs, and significantly increased transforming growth factor-β (TGF-β), IL-10, IL-6 and PGE2 levels. Furthermore, MVs decreased the number of Th17 cells and the levels of IL-17, and increased FoxP3+ regulatory T cells in GAD65-stimulated PBMCs.Conclusions/interpretationThese results provide evidence that MSC-derived MVs can inhibit in vitro a proinflammatory response to an islet antigenic stimulus in type 1 diabetes. The action of MVs involves PGE2 and TGF-β signalling pathways and IL-10 secretion, suggesting a switch to an anti-inflammatory response of T cells.

Human mesenchymal stem cells modulate cellular immune response to islet antigen glutamic acid decarboxylase in type 1 diabetes.

CONTEXT Mesenchymal stem cells (MSCs) exert an immunosuppressive effect on the immune system. However, studies on the immunomodulatory potential of MSCs in type 1 diabetes are lacking. OBJECTIVE We aimed to evaluate whether human MSCs may inhibit in vitro pancreatic islet antigen-specific T cell activation in type 1 diabetes. DESIGN Human MSCs were isolated and characterized. Peripheral blood mononuclear cells (PBMCs) were obtained from nine type 1 diabetic patients at disease onset and 13 healthy control subjects. IFN-gamma, IL-10, and IL-4 enzyme-linked immunospot responses of lymphocytes incubated with glutamic acid decarboxylase 65 (GAD65) were investigated in PBMC cultures and PBMC/MSC cocultures. Levels of prostaglandin E2 (PGE2), IFN-gamma, IL-4, and IL-10 in supernatants were measured by ELISA. PGE2 inhibition experiments with NS-398 and indomethacin were also performed. RESULTS Five diabetic patients were identified with a positive PBMC IFN-gamma response to GAD65 and negative IL-10 and IL-4 response. PBMC/MSC cocultures resulted in a significant decrease in the number of spots and in detection of IL-4-secreting cells. PGE2 inhibitors abrogated the immune-suppressive effect, indicating an involvement of PGE2 production, and the constitutive production of PGE2 by MSCs was enhanced in PBMC/MSC coculture. Moreover, in GAD-responder patients, GAD-stimulated PBMC/MSC cocultures significantly decreased secretion of IFN-gamma and IL-10 and increased secretion of IL-4. CONCLUSIONS These results provide evidence that human MSCs abrogate in vitro a proinflammatory T helper type 1 response to an islet antigenic stimulus in type 1 diabetes. MSCs induce IL-4-producing cells, suggesting a possible switch to an antiinflammatory T helper type 2 signaling of T cells.

CCL16/LEC powerfully triggers effector and antigen- presenting functions of macrophages and enhances T cell cytotoxicity

The huan CC chemokine CCL16, a liver‐expressed chemokine, enhances the killing activity of mouse peritoneal macrophages by triggering their expression of tumor necrosis factor α (TNF‐α) and Fas ligand. Macrophages also respond to CCL16 by enhancing their production of monocyte chemoattractant protein‐1, regulated on activation, normal T cells expressed and secreted chemokines, and interleukin (IL)‐1β, TNF‐α, and IL‐12. The effect of CCL16 is almost as strong as that of lipopolysaccharide and interferon‐γ, two of the best macrophage activators. Moreover, CCL16‐activated macrophages overexpress membrane CD80, CD86, and CD40 costimulatory molecules and extensively phagocytose tumor cell debris. On exposure to such debris, they activate a strong, tumor‐specific, cytolytic response in virgin T cells. Furthermore, cytolytic T cells generated in the presence of CCL16 display a higher cytotoxicity and activate caspase‐8 in tumor target cells. This ability to activate caspase‐8 depends on their overexpression of TNF‐α and Fas ligand induced by CCL16. These data reveal a new function for CCL16 in the immune‐response scenario. CCL16 significantly enhances the effector and the antigen‐presenting function of macrophages and augments T cell lytic activity.

IL-10 enhances CCL2 release and chemotaxis induced by CCL16 in human monocytes

CCL16 is a CC chemokine originally identified as a liver-expressed chemokine. Its expression has been detected in activated monocytes where it is up-regulated by stimulation with IL-10. This is in contrast with IL-10s inhibition of the expression of most chemokines. CCL16 is chemotactic for monocytes, lymphocyte and dendritic cells. We investigated whether CCL16 displays biological activities other than chemotaxis and whether IL-10 affects monocyte response to CCL16. We show that CCL16 induces the expression of CCL2 at the mRNA and protein level, but does not affect that of CCL5, CCL18 and proinflammatory cytokines. This effect was prevented by treatment with pertussis toxin and may thus be mediated by G-protein-coupled receptors. IL-10 markedly increased CCL2 production induced by CCL16, but suppressed that of CXCL8. It also enhanced the chemotactic response to CCL16. Addition of antibodies blocking CCR1, but not CCR8, prevented this enhanced chemotactic response and suggested that CCR1 is primarily involved. We propose that IL-10 modulates the effects of CCL16 on monocytes by increasing their CCR1-dependent response. The coordinated secretion of CCL16 and IL-10 may thus enhance monocyte infiltration.

CC-chemokine ligand 16 induces a novel maturation program in human immature monocyte-derived dendritic cells

Dendritic cells (DCs) are indispensable for initiation of primary T cell responses and a host’s defense against infection. Many proinflammatory stimuli induce DCs to mature (mDCs), but little is known about the ability of chemokines to modulate their maturation. In the present study, we report that CCL16 is a potent maturation factor for monocyte-derived DCs (MoDCs) through differential use of its four receptors and an indirect regulator of Th cell differentiation. MoDCs induced to mature by CCL16 are characterized by increased expression of CD80 and CD86, MHC class II molecules, and ex novo expression of CD83 and CCR7. They produce many chemokines to attract monocytes and T cells and are also strong stimulators in activating allogeneic T cells to skew toward Th1 differentiation. Interestingly, they are still able to take up Ag and express chemokine receptors usually bound by inflammatory ligands and can be induced to migrate to different sites where they capture Ags. Our findings indicate that induction of MoDC maturation is an important property of CCL16 and suggest that chemokines may not only organize the migration of MoDCs, but also directly regulate their ability to prime T cell responses.

Human mesenchymal stem cells and derived extracellular vesicles induce regulatory dendritic cells in type 1 diabetic patients

Aims/hypothesisMesenchymal stem cells (MSCs) can exert an immunosuppressive effect on any component of the immune system, including dendritic cells (DCs), by direct contact, the release of soluble markers and extracellular vesicles (EVs). We evaluated whether MSCs and MSC-derived EVs have an immunomodulatory effect on monocyte-derived DCs in type 1 diabetes.MethodsBone marrow derived MSCs were characterised and EVs were obtained by ultracentrifugation. DCs were differentiated from CD14+ cells, obtained from nine type 1 diabetic patients at disease onset, pulsed with antigen GAD65 and cultured with MSCs or EVs. Levels of DC maturation and activation markers were evaluated by flow cytometry. GAD65-pulsed DCs and autologous CD14− cell were co-cultured and IFN-γ enzyme-linked immunosorbent spot responses were assayed. Secreted cytokine levels were measured and Th17 and regulatory T cells were analysed.ResultsMSC- and EV-conditioned DCs acquired an immature phenotype with reduced levels of activation markers and increased IL-10 and IL-6 production. Conditioned DC plus T cell co-cultures showed significantly decreased IFN-γ spots and secretion levels. Moreover, higher levels of TGF-β, IL-10 and IL-6 were detected compared with unconditioned DC plus T cell co-cultures. Conditioned DCs decreased Th17 cell numbers and IL-17 levels, and increased FOXP3+ regulatory T cell numbers. EVs were internalised by DCs and EV-conditioned DCs exhibited a similar effect.Conclusions/interpretationIn type 1 diabetes, MSCs induce immature IL-10-secreting DCs in vitro, thus potentially intercepting the priming and amplification of autoreactive T cells in tissue inflammation. These DCs can contribute to the inhibition of inflammatory T cell responses to islet antigens and the promotion of the anti-inflammatory, regulatory responses exerted by MSCs.

Chimeric Rat/Human HER2 Efficiently Circumvents HER2 Tolerance in Cancer Patients

Purpose: Despite the great success of HER2 vaccine strategies in animal models, effective clinical results have not yet been obtained. We studied the feasibility of using DNA coding for chimeric rat/human HER2 as a tool to break the unresponsiveness of T cells from patients with HER2-overexpressing tumors (HER2-CP). Experimental Design: Dendritic cells (DCs) generated from patients with HER2-overexpressing breast (n = 28) and pancreatic (n = 16) cancer were transfected with DNA plasmids that express human HER2 or heterologous rat sequences in separate plasmids or as chimeric constructs encoding rat/human HER2 fusion proteins and used to activate autologous T cells. Activation was evaluated by IFN-γ ELISPOT assay, perforin expression, and ability to halt HER2+ tumor growth in vivo. Results: Specific sustained proliferation and IFN-γ production by CD4 and CD8 T cells from HER2-CP was observed after stimulation with autologous DCs transfected with chimeric rat/human HER2 plasmids. Instead, T cells from healthy donors (n = 22) could be easily stimulated with autologous DCs transfected with any human, rat, or chimeric rat/human HER2 plasmid. Chimeric HER2-transfected DCs from HER2-CP were also able to induce a sustained T-cell response that significantly hindered the in vivo growth of HER2+ tumors. The efficacy of chimeric plasmids in overcoming tumor-induced T-cell dysfunction relies on their ability to circumvent suppressor effects exerted by regulatory T cells (Treg) and/or interleukin (IL)-10 and TGF-β1. Conclusions: These results provide the proof of concept that chimeric rat/human HER2 plasmids can be used as effective vaccines for any HER2-CP with the advantage of being not limited to specific MHC. Clin Cancer Res; 20(11); 2910–21. ©2014 AACR.

Lack of Plasma Protein Hemopexin Dampens Mercury-Induced Autoimmune Response in Mice

Several factors affect the autoimmune response, including iron-dependent modulation of T cells. Hemopexin is the plasma protein with the highest binding affinity to heme. It mediates heme-iron recovery in the liver, thus controlling heme-iron availability in peripheral cells. The aim of the present study was to investigate the role of hemopexin in the progress of an autoimmune response. To this end, we chose a mouse model of mercury-induced autoimmunity and evaluated the susceptibility of hemopexin-null mice to mercury treatment compared with wild-type controls. In this study we show that lack of hemopexin dampens mercury-induced autoimmune responses in mice. Hemopexin-null mice produced fewer antinuclear autoantibodies and had reduced deposits of immune complexes in the kidney after mercuric chloride treatment compared with wild-type mice. These features were associated with a reduction in activated T cells and lower absolute B cell number in spleen and impaired IgG1 and IgG2a production. In contrast, in hemopexin-null mice the response to OVA/CFA immunization was maintained. In addition, hemopexin-null mice had reduced transferrin receptor 1 expression in T cells, possibly due to the increase in heme-derived iron. Interestingly, CD4+T cells isolated from mercury-treated hemopexin-null mice show reduced IFN-γ-dependent STAT1 phosphorylation compared with that of wild-type mice. Our data suggest that hemopexin, by controlling heme-iron availability in lymphocytes, modulates responsiveness to IFN-γ and, hence, autoimmune responses.

CCL16 enhances the CD8+ and CD4+ T cell reactivity to human HER-2 elicited by dendritic cells loaded with rat ortholog HER-2.

T cells from HLA A2+ healthy donors were co-cultured with autologous dendritic cells (DC) loaded with apoptotic tumor cells expressing rat neu, and were induced to mature by tumor necrosis factor (TNF)α and interleukin (IL)-1β (mDC neu ) or by the CCL16 chemokine (CCL16/mDC neu ). Priming by CCL16/mDC neu induces a larger population of T cells that express cytoplasmatic interferon (IFN)γ, TNFα, perforin and granzyme B compared to those primed by mDCneu. T cells primed by CCL16/mDC neu release IFNγ in response to human HER-2+ cells and kill human HER-2+ target cells more efficiently than those primed by mDCneu. Our results show that both the loading of DC with xenogeneic rat neu and their maturation by CCL16 are two issues of critical importance for the elicitation of an effective response to human HER-2 in T cells from normal donors.