Biomedical subjects

Giovanna Lombardi

Publications and source records attributed to Giovanna Lombardi.

At least 19 recordsLinked to original sources

PubMed2007

Mesenchymal stem cells inhibit dendritic cell differentiation and function by preventing entry into the cell cycle.

BACKGROUND: Mesenchymal stem cells (MSCs) play a crucial role in hematopoietic development and have been shown to exert a powerful immunosuppressive effect. In this study, we investigated the effect of bone marrow MSC on the differentiation and function of peripheral blood monocytes into dendritic cells (DCs). METHODS: Human MSCs, generated from normal bone marrow, were added to peripheral blood monocytes stimulated in vitro with granulocyte-macrophage colony stimulating factor and interleukin-4 to become DCs. Monocytes were then examined for the expression of markers characteristic of DCs and their ability to stimulate allogeneic T cells. In addition, the effect of MSCs on the cell cycle of monocyte-derived DCs and the expression of various cell cycle proteins were analyzed by cytometric analysis and Western blotting with specific antibodies. RESULTS: MSCs blocked the differentiation of monocytes into DCs and impaired their antigen-presenting ability. This resulted from a block of monocytes from entering the G1 phase of the cell cycle with a progressive number of cells accumulating in the G0 phase. Cyclin D2 was downregulated. However, differently from what was observed in T-cells stimulated in the presence of MSCs, the expression of p27 was found decreased, suggesting the involvement of similar but not identical pathways. CONCLUSIONS: We conclude that MSCs impair monocyte differentiation and function by interfering with the cell cycle. These findings imply that MSC-induced immunosuppression might be a side product of a more general antiproliferative effect.

Antigens, CD↗

PubMed2006

Generation and expansion of human CD4+ CD25+ regulatory T cells with indirect allospecificity: Potential reagents to promote donor-specific transplantation tolerance.

BACKGROUND: Harnessing naturally arising CD4+ CD25+ regulatory T cells (Tregs) for potential adoptive cell therapy is hampered by their innate autoreactivity and their limited number. METHODS: CD4+ CD25+ Tregs were purified from peripheral blood of human leukocyte antigen (HLA) DR1*0101+ A2- individuals, and stimulated with autologous monocyte-derived dendritic cells (DCs). RESULTS: Here we show that CD4+ CD25+ Tregs specific for an HLA A2 (103-120) peptide can be selected from the peripheral blood CD4+ CD25+ T cell population of a healthy individual and detected using a tetramer comprised of HLA DRB1*0101 and the A2 peptide. The selected cells can be expanded substantially (i.e., a 1600-fold increase over a two-week period) by T-cell receptor (TCR) stimulation and high-doses of interleukin-2 (IL-2). The CD4+ CD25+Tregs with indirect allospecificity for the A2 peptide showed more potent antigen-specific suppression than polyclonal CD4+ CD25+ Tregs. CONCLUSIONS: These data may pave the way for clinical studies using CD4+ CD25+ Tregs with indirect allospecificity as therapeutic reagents for the induction of donor-specific transplantation tolerance.

Animals↗

PubMed2006

Aspirin modified dendritic cells are potent inducers of allo-specific regulatory T-cells.

Salicylic acid (aspirin) is a widely used pharmacological agent with immunodmodulatory properties. Dendritic cells are key regulators of the immune response, and are capable of inducing hyporesponsiveness and regulatory activity in CD4+ T-cells. We have demonstrated that aspirin-treated dendritic cells are effective at antigen processing and presentation, and possess a unique potency for inducing regulatory activity in responder T-cells. Unlike immature dendritic cells, aspirin DCs are resistant to the effects of maturational stimuli, as determined by low levels of CD40, CD80, CD83 and CD86 expression. Aspirin DCs were demonstrated to express high levels of the co-inhibitor of T-cell activation ILT-3. Aspirin DCs themselves produce less IL-10 and more IL-12 than immature DCs, but no specific cytokine is necessary for their tolerogenic capacity. When naïve CD4+ T-cells are exposed to aspirin DCs they produce significant levels of IFNgamma but these same T-cells are hypo-proliferative. Aspirin-treated DCs demonstrate the characteristics of a potential immunotherapy for controlling unwanted immune-responses such as the indirect pathway of allo-recognition that drives chronic allograft rejection.

Antigens, CD↗

PubMed2006

Chronic exposure to Helicobacter pylori impairs dendritic cell function and inhibits Th1 development.

Helicobacter pylori causes chronic gastric infection that affects the majority of the world's population. Despite generating an inflammatory response, the immune system usually fails to clear the infection. Since dendritic cells (DCs) play a pivotal role in shaping the immune response, we investigated the effects of H. pylori on DC function. We have demonstrated that H. pylori increased the expression of activation markers on DCs while upregulating the inhibitory B7 family molecule, PD-L1. Functionally, H. pylori-treated DCs resulted in the production of interleukin-10 (IL-10) and IL-23 but not of alpha interferon (IFN-alpha). While very little or no IL-12 was produced to H. pylori alone, simultaneous ligation of CD40 on DCs induced IL-12 release. We also demonstrated that DCs treated with H. pylori-induced IFN-gamma production by allogeneic naive T cells. However, stimulation of DCs with H. pylori for an extended period of time impaired their ability to produce cytokines after CD40 ligation and limited their ability to promote IFN-gamma release, suggesting that the DCs had become exhausted by the prolonged stimulation. The effect of chronic infection with H. pylori on DC function was further investigated by focusing on DC development. Demonstrating that monocytes differentiated into DCs in the presence of H. pylori exhibited an exhausted phenotype with an impaired ability to produce IL-12 and a downregulation of CD1a. Our results raise the possibility that in chronic H. pylori infection DCs become exhausted after prolonged antigen exposure leading to suboptimal Th1 development. This effect may contribute to persistence of H. pylori infection.

Antigens, CD↗

PubMed2006

New trends in immunosuppression and immunotherapy.

In the past years, research on immune regulation has exploded thanks to the re-emergence of T suppressor cells (Ts), now coined as regulatory T cells (Tregs). It has been established that different populations of Tregs exist in the immune system, and the generation, mechanism of suppression and the cellular and molecular targets of Tregs are being unraveled. This will lead to new opportunities for immune intervention to treat autoimmune disease and to achieve clinical transplantation tolerance. The scientific progress in the field of translational medicine was presented at the 7th International Conference on New Trends in Immunosuppression and Immunotherapy in Berlin in February 2006. In this special issue of International Immunopharmacology, several papers submitted to this congress will highlight some of the advances in this burgeoning field of translational medicine.

Adoptive Transfer↗

PubMed2006

In-vitro generation and characterisation of murine CD4+CD25+ regulatory T cells with indirect allospecificity.

Naturally arising CD4(+)CD25(+) regulatory T cells play a pivotal role in the prevention of autoimmunity and in the induction of donor-specific transplantation tolerance. Harnessing regulatory cells for potential adoptive cell therapy is hampered by their lack of antigen-specificity and their limited numbers. Here we describe the generation and expansion of murine CD4(+)CD25(+) T cells with antigen-specificity for an K(d) peptide as potential reagents for adoptive cell therapy in promoting donor-specific transplantation tolerance. Using bone marrow-derived autologous dendritic cells pulsed with the K(d) peptide, we generated T cell lines from purified CD4(+)CD25(+) T cells from C56BL/6 mice. The T cell lines expressed high level of CD25 and low level of CD45RB and CD69. They maintained the expression of CD62L, GITR, CTLA-4 and more importantly FoxP3. The CD4(+)CD25(+) T cell lines were anergic after TCR stimulation and produced little cytokine such as IL-2 and IFN-gamma. Importantly, they were more potent than freshly isolated CD4(+)CD25(+) T cells in suppressing proliferation and cytokine secretion by effector CD4(+) T cells. Furthermore, the CD4(+)CD25(+) T cell lines could be expanded to large cell numbers and maintained in culture up to 1 year. The K(d)-specific CD4(+)CD25(+) T cell lines will be invaluable in devising a strategy for the induction of cardiac transplantation tolerance in wild-type B6 mice carrying a full mismatch BALB/c heart.

Animals↗

PubMed2006

In vitro expanded alloantigen-specific CD4+CD25+ regulatory T cell treatment for the induction of donor-specific transplantation tolerance.

The key goal in clinical transplantation is the induction of donor-specific transplantation tolerance to minimise the morbidity and mortality associated with long-term immunosuppression. Naturally occurring CD4(+)CD25(+) regulatory T cells (Tregs) expressing forkhead transcript factor FoxP3 play a crucial role in the prevention of autoimmunity, and appear to mediate transplantation tolerance, and these cells can have indirect allospecificity for donor antigens. Here we show that self-reactive human CD4(+)CD25(+) Tregs can be subverted into allopeptide-specific cells in vitro and be expanded to large cell numbers, and that similar in vitro expanded murine CD4(+)CD25(+) Tregs with indirect allospecificity were capable of inducing donor-specific experimental transplantation tolerance. These data provide a platform for clinical studies using CD4(+)CD25(+) Tregs with indirect allospecificity as potential reagents for the induction of donor-specific transplantation tolerance.

Adoptive Transfer↗

PubMed2006

A novel pathway of antigen presentation by dendritic and endothelial cells: Implications for allorecognition and infectious diseases.

Dendritic cells (DCs) are the major antigen presenting cells capable of stimulating T cell responses following either organ transplantation or a viral infection. In the context of allorecognition, T cells can be activated following presentation of alloantigens by donor DCs (direct), as well as by recipient DCs presenting processed donor major histocompatibility complex (MHC) as peptides (indirect). We have recently described another mechanism by which alloreactive T cells are activated. Recipient DCs can acquire donor MHC through cell-to-cell contact and this acquired MHC can stimulate a T cell response (the semidirect pathway). Similarly, during a viral infection, DCs are capable of stimulating T cells directly, as occurs when infected DCs present processed viral antigens, or indirectly by a process known as cross-presentation. Although cross-presentation of exogenous antigen is an important mechanism for controlling infectious diseases, it is possible that peptide:MHC acquisition (the semidirect pathway) may also play a part in immunity against pathogens. In this review, we discuss the possible contributions of the semidirect pathway/MHC transfer in infectious disease.

Antigen Presentation↗

PubMed2006

Altered proximal T cell receptor (TCR) signaling in human CD4+CD25+ regulatory T cells.

CD4+CD25+ regulatory T cells play an important role in peripheral tolerance. Upon T cell receptor (TCR)-mediated activation, the cells fail to proliferate but are induced to have a suppressor function. The intracellular signaling events that lead to their responses have not been elucidated. In this study, we have examined the proximal TCR signaling events in freshly isolated human CD4+CD25+ regulatory T cells after TCR ligation. In contrast to CD4+CD25- T cells, TCR ligation of CD4+CD25+ regulatory T cells by anti-CD3 cross-linking resulted in a lower calcium influx and extracellular signal-regulated kinase 1/2 phosphorylation. Examination of the CD3zeta chain phosphorylation status indicated that CD4+CD25+ regulatory T cells have poor phosphorylation of the protein and consequently, reduced recruitment of zeta-associated protein-70 to the TCR immunoreceptor tyrosine motif. The adaptor protein, Src homology 2 domain-containing leukocyte phosphoprotein of 76 kDa, which relays signals to downstream signaling components, also showed reduced phosphorylation, which correlated with reduced VAV guanine nucleotide exchange factors association. Consistent with other findings, the defect is accompanied with impaired actin cap formation, implicating a failure of actin remodeling of the cells. Together, our results demonstrate that CD4+CD25+ regulatory T cells have altered TCR proximal signaling pathways, which could be critical for inducing the distinct behavior of these cells.

Actins↗

PubMed2006

Modulation of dendritic cell phenotype and function in an in vitro model of the intestinal epithelium.

A network of dendritic cells (DC) can be detected in close proximity to the epithelial cells overlying Peyer's patches in the gut. Intestinal DC show distinct phenotypes as compared to DC from the systemic lymph nodes (relatively low MHC and costimulatory molecules and high IL-10 and TGFbeta) and may play a role in maintaining tolerance to enteric antigens. We show that a similar phenotype is induced in the presence of a polarised epithelial cell monolayer in vitro. Monocyte-derived DC were co-cultured with Caco-2 intestinal epithelial monolayers for 24 h. Co-culture resulted in DC with reduced expression of MHC class II, CD86, and CD80, and poor T cell stimulatory capacity. Cytokine profiles showed reduced levels of inflammatory cytokine production, and co-cultured DC were less sensitive to stimulation via Toll-like receptors (TLR2, 4, and 6) as a result of increased levels of autocrine TGFbeta production. However, phenotypic changes in co-cultured DC could not be blocked by removal of apoptotic cells or addition of anti-TGFbeta antibodies, suggesting that other soluble factors are involved in DC modulation. Thus, polarised epithelial cell monolayers create a 'tolerogenic' environment which modulates the activity of DC. These results highlight the regulatory importance of the epithelial microenvironment at mucosal surfaces.

Antigens, CD↗

PubMed2006

Helicobacter pylori has stimulatory effects on naive T cells.

BACKGROUND: Despite an apparently active host response, Helicobacter pylori infection can persist for life. Unexpectedly, T cells from apparently uninfected individuals respond to H. pylori antigen by proliferating. Also, the T-cell proliferative response appears to be less in infected compared with uninfected individuals. MATERIALS AND METHODS: We have investigated the T-cell response of isolated human peripheral blood, naive, and memory CD4+ T cells to H. pylori antigen in infected and uninfected subjects. RESULTS: In agreement with previous findings, the peripheral blood proliferative response was higher in uninfected compared with infected subjects. Interestingly, there was a response in CD4+ CD45RO+ (memory) and CD4+CD45RA+ (naive) subsets. The RO/RA ratio of the response to H. pylori antigen was 0.8-2.1 in both H. pylori-positive and H. pylori-negative subjects, which was similar to that of a known superantigen (2.5 and 2.2 in Helicobacter-positive and -negative subjects, respectively) whereas the RO/RA response ratio to a recall antigen (tetanus toxoid) was 9.8 and 18.7 in Helicobacter-positive and -negative subjects, respectively. Mononuclear cells isolated from cord blood also responded to H. pylori antigen, whereas there was no response to tetanus toxoid. The cord blood response and CD4+ CD45RA+ cell response to H. pylori antigen were inhibited predominantly by anti-HLA-DR and to some extent by anti-HLA-DQ antibodies. Investigation of the response to five different recombinant H. pylori antigens identified two that produced a response in naive T cells. CONCLUSIONS: These data suggest that H. pylori possesses molecules that cause higher than expected proliferation of naive T cells.

Antibodies, Monoclonal↗

PubMed2005

Secretion of IFN-gamma and not IL-2 by anergic human T cells correlates with assembly of an immature immune synapse.

We report differences in the supramolecular organization of the immunologic synapse (IS) formed by resting and anergic human T cells with agonist peptide-loaded antigen-presenting cells (APCs). T cells reactive to influenza A hemagglutinin peptide or Fel d 1 peptide 4 were rendered both anergic and regulatory by incubation with high doses of agonist peptide in the absence of APCs. At the IS between resting T cells and peptide-loaded APCs, both CD3epsilon and CD3zeta initially accumulate within a ring or arc before redistributing within 30 minutes to single or multiple foci more central to the contact. In contrast, at synapses formed by anergized T cells, CD3epsilon and CD3zeta remained organized within an arc or ring and failed to redistribute centrally. However, intercellular communication between anergic human T cells and agonist peptide-loaded APCs was not a null event, since it triggered secretion of T-cell interferon gamma (IFN-gamma) but not, for example, interleukin 2 (IL-2). Thus, distinct organizations of CD3 at the T-cell IS correlate with different cytokine profiles; the mature IS formed by resting T cells correlates with their production of both IFN-gamma and IL-2, whereas the immature IS formed by anergic T cells seems able to facilitate IFN-gamma but not IL-2 production.

Antigen-Presenting Cells↗

PubMed2005

Creation of tolerogenic human dendritic cells via intracellular CTLA4: a novel strategy with potential in clinical immunosuppression.

Activation of T lymphocytes requires the recognition of peptide-major histocompatibility complexes (MHCs) and costimulatory signals provided by antigen-presenting cells (APCs). It has been shown that T-cell activation without costimulation can lead to anergy. In this study, we developed a novel strategy to inhibit expression of B7 molecules (CD80/86) by transfecting APCs with a gene construct encoding a modified cytotoxic T lymphocyte antigen 4 (CTLA4) molecule (CTLA4-KDEL) that is targeted to the endoplasmic reticulum (ER). APCs expressing this construct failed to express CD80/86 on their surface, were unable to stimulate allogeneic and peptide-specific T-cell responses, and induced antigen-specific anergy of the responding T cells. Cells expressing CTLA4-KDEL do not up-regulate the indoleamine 2, 3-dioxygenase enzyme, unlike cells treated with soluble CTLA4-immunoglobin (Ig). This gene-based strategy to knock out surface receptors is an attractive alternative to using immature dendritic cells for preventing transplant rejection and treating of autoimmune diseases.

Antigens, CD↗

PubMed2005

Inhibition of NF-kappa B and oxidative pathways in human dendritic cells by antioxidative vitamins generates regulatory T cells.

Dendritic cells (DCs) are central to T cell immunity, and many strategies have been used to manipulate DCs to modify immune responses. We investigated the effects of antioxidants ascorbate (vitamin C) and alpha-tocopherol (vitamin E) on DC phenotype and function. Vitamins C and E are both antioxidants, and concurrent use results in a nonadditive activity. We have demonstrated that DC treated with these antioxidants are resistant to phenotypic and functional changes following stimulation with proinflammatory cytokines. Following treatment, the levels of intracellular oxygen radical species were reduced, and the protein kinase RNA-regulated, eukaryotic translation initiation factor 2alpha, NF-kappaB, protein kinase C, and p38 MAPK pathways could not be activated following inflammatory agent stimulation. We went on to show that allogeneic T cells (including CD4(+)CD45RO, CD4(+)CD45RA, and CD4(+)CD25(-) subsets) were anergized following exposure to vitamin-treated DCs, and secreted higher levels of Th2 cytokines and IL-10 than cells incubated with control DCs. These anergic T cells act as regulatory T cells in a contact-dependent manner that is not dependent on IL-4, IL-5, IL-10, IL-13, and TGF-beta. These data indicate that vitamin C- and E-treated DC might be useful for the induction of tolerance to allo- or autoantigens.

Antioxidants↗

PubMed2005

Modulation of human dendritic-cell function following transduction with viral vectors: implications for gene therapy.

Genetic modification of dendritic-cell (DC) function is an attractive approach to treat disease, either using mature DCs (mDCs) to immunize patients, or immature DCs (iDCs) to induce tolerance. Viral vectors are efficient at transducing DCs, and we have investigated the effect of transduction with a variety of viral vectors on the phenotype and function of DCs. Adenovirus (Ad), human immunodeficiency virus (HIV), equine anemia virus (EIAV), and Moloney murine leukemia virus (MMLV) all up-regulate costimulatory molecules and major histocompatibility complex (MHC) class II expression on DCs, as well as, in the case of Ad and lentiviral vectors, inducing production of Th1 and proinflammatory cytokines. Following transduction there is activation of double-stranded (ds) RNA-triggered pathways resulting in interferon (IFN) alpha/beta production. In addition, the function of virally infected DCs is altered; iDCs have an increased, and mDCs a decreased, ability to stimulate a mixed lymphocyte reaction (MLR). Viral transduction of mDCs results in up-regulation of the indoleamine 2,3-dioxygenase (IDO) enzyme, which down-regulates T-cell responsiveness. Inhibition of IDO restores the ability of mDCs to stimulate an MLR, indicating that IDO is responsible for the modulation of mDC function. These data have important implications for the use of viral vectors in the transduction of DCs.

Adenoviridae↗

PubMed2005

Type I interferons and the innate immune response--more than just antiviral cytokines.

The role of type I interferon (referred to as IFN in this review) in early antiviral immunity is well known. More recently IFN has been shown to be a potent regulator of adaptive immunity. It is now becoming clear that a broad range of viruses, bacteria and even parasites express ligands capable of stimulating a growing number of signalling pathways that results in, often subtype specific, induction of IFN. Of particular interest are the signalling pathways associated with the Toll-like receptors. This family of receptors, each able to induce signals in response to a variety of ligands, initiates the pro-inflammatory response. They also contain members that have the capacity to induce IFN, making use of, and perhaps promoting the evolution of its pleiotropic responses. Greater knowledge of the events that result in induction of IFN is necessary in understanding the specificity of expression of an increasingly complex and important aspect of our immune system. This may reveal to us further therapeutic opportunities, either in the use of IFN or in the manipulation of their expression. This review details the established knowledge and recent advances made in understanding how and under what circumstances the IFNs are expressed, starting with brief overviews of IFN and Toll-like receptors before following the molecular processes from induction of IFN, activation of the JAK-STAT pathway and finally the expression of interferon stimulated genes and their functions.

Animals↗

PubMed2005

Activated CD1d-restricted natural killer T cells secrete IL-2: innate help for CD4+CD25+ regulatory T cells?

CD4(+)CD25(+) and CD1d-restricted natural killer T (NKT) cells are thymus-derived self-reactive regulatory T cells that play a key role in the control of pathological immune responses. Little is known about functional cooperation between innate regulatory NKT cells and adaptive CD4(+)CD25(+) regulatory cells. Here we show that human CD4(+)Valpha24(+)Vbeta11(+) (CD4(+) NKT) cells isolated from peripheral blood by flow cytometric cell sorting secrete substantial amounts of IL-2 after stimulation with dendritic cells (DC) and alpha-Galactosylceramide. When cocultured with CD4(+)CD25(+) cells, CD4(+) NKT cells promoted moderate proliferation of CD4(+)CD25(+) cells. The proliferation of CD4(+)CD25(+) T cells was due to soluble IL-2 produced by activated CD4(+) NKT cells. The expanded CD4(+)CD25(+) cells remained anergic and retained their potent suppressive properties. These findings indicate that unlike conventional CD4(+) and CD8(+) T cells, which are susceptible to CD4(+)CD25(+) regulatory cell suppression, NKT cells promote CD4(+)CD25(+) regulatory cell proliferation. These data raise the possibility that NKT cells can function as helper cells to CD4(+)CD25(+) regulatory T cells, thereby providing a link between the two naturally occurring populations of regulatory T cells.

Antigens, CD1↗

PubMed2005

Distinct effects of CD86-mediated costimulation on resting versus activated human CD4+ T cells.

CD80 and CD86 are important in the initiation of T cell immunity. Although their costimulatory function has long been appreciated, it remains unclear whether the biological significance of the two B7 isoforms resides in their different patterns and kinetics of expression or whether differences exist in their function. We have addressed this issue using HLA-DR1 transfectants co-expressing CD80, CD86, or both molecules as stimulators for naïve, memory, and activated human CD4+ T cells. Both CD80 and CD86 efficiently costimulated alloresponses by unseparated peripheral blood CD4+ T cells; however, CD86 was substantially inferior in costimulating alloresponses by separated memory T cells, and completely incompetent in costimulating three human T cell clones. Furthermore, CD80/CD86 double transfectants stimulated lower responses by the clones than cells expressing CD80 alone. That CD86 was actively inhibitory rather than merely neutral was evidenced by the increase in response to the double CD80/CD86 APC when anti-CD86 antibody was added. Furthermore, addition of anti-CTLA-4 Fab to cultures of HLA-DR1 transfectants co-expressing CD86, fully restored the proliferative response. These results indicate that CD80 and CD86 mediate distinct signals in previously activated T cells, and demonstrate that CTLA-4 ligation may dominate the outcome of CD86-mediated costimulation of activated CD4+ T cells.

Antigens, CD↗