Federica M. Marelli-Berg

Characterization and Clinical Application of Human CD34+ Stem/Progenitor Cell Populations Mobilized into the Blood by Granulocyte Colony‐Stimulating Factor

A phase I study was performed to determine the safety and tolerability of injecting autologous CD34+ cells into five patients with liver insufficiency. The study was based on the hypothesis that the CD34+ cell population in granulocyte colony‐stimulating factor (G‐CSF)‐mobilized blood contains a subpopulation of cells with the potential for regenerating damaged tissue. We separated a candidate CD34+ stem cell population from the majority of the CD34+ cells (99%) by adherence to tissue culture plastic. The adherent and nonadherent CD34+ cells were distinct in morphology, immunophenotype, and gene expression profile. Reverse transcription‐polymerase chain reaction‐based gene expression analysis indicated that the adherent CD34+ cells had the potential to express determinants consistent with liver, pancreas, heart, muscle, and nerve cell differentiation as well as hematopoiesis. Overall, the characteristics of the adherent CD34+ cells identify them as a separate putative stem/progenitor cell population. In culture, they produced a population of cells exhibiting diverse morphologies and expressing genes corresponding to multiple tissue types. Encouraged by this evidence that the CD34+ cell population contains cells with the potential to form hepatocyte‐like cells, we gave G‐CSF to five patients with liver insufficiency to mobilize their stem cells for collection by leukapheresis. Between 1 × 106 and 2 × 108 CD34+ cells were injected into the portal vein (three patients) or hepatic artery (two patients). No complications or specific side effects related to the procedure were observed. Three of the five patients showed improvement in serum bilirubin and four of five in serum albumin. These observations warrant further clinical trials.

A Novel Pathway of Alloantigen Presentation by Dendritic Cells

In the context of transplantation, dendritic cells (DCs) can sensitize alloreactive T cells via two pathways. The direct pathway is initiated by donor DCs presenting intact donor MHC molecules. The indirect pathway results from recipient DCs processing and presenting donor MHC as peptide. This simple dichotomy suggests that T cells with direct and indirect allospecificity cannot cross-regulate each other because distinct APCs are involved. In this study we describe a third, semidirect pathway of MHC alloantigen presentation by DCs that challenges this conclusion. Mouse DCs, when cocultured with allogeneic DCs or endothelial cells, acquired substantial levels of class I and class II MHC:peptide complexes in a temperature- and energy-dependent manner. Most importantly, DCs acquired allogeneic MHC in vivo upon migration to regional lymph nodes. The acquired MHC molecules were detected by Ab staining and induced proliferation of Ag-specific T cells in vitro. These data suggest that recipient DCs, due to acquisition of donor MHC molecules, may link T cells with direct and indirect allospecificity.

Isolation of endothelial cells from murine tissue

The isolation and long-term culture of murine endothelial cells (ECs) has often proven a difficult task. In this paper we describe a quick, efficient protocol for the isolation of microvascular endothelial cells from murine tissues. Murine lung or heart are mechanically minced and enzymatically digested with collagenase and trypsin. The single cell suspension obtained is then incubated with an anti-CD31 antibody, anti-CD105 antibody and with biotinylated isolectin B-4. Pure EC populations are finally obtained by magnetic bead separation using rat anti-mouse Ig- and streptavidin-conjugated microbeads. EC cultures are subsequently expanded and characterised. The surface molecule expression by the primary cultures of murine EC obtained from lung and heart tissue is analysed and compared to that of a murine endothelioma and of primary cultures of murine renal tubular epithelial cells. The phenotype and morphology of these cultures remain stable over 10-15 passages in culture, and no overgrowth of contaminating cells of non-endothelial origin is observed at any stage.

Membrane phosphatidylserine distribution as a non-apoptotic signalling mechanism in lymphocytes

Phosphatidylserine (PS) exposure is normally associated with apoptosis and the removal of dying cells. We observed that PS is exposed constitutively at high levels on T lymphocytes that express low levels of the transmembrane tyrosine phosphatase CD45RB. CD45 was shown to be a negative regulator of PS translocation in response to various signals, including activation of the ATP receptor P2X7. Changes in PS distribution were shown to modulate several membrane activities: Ca2+ and Na+ uptake through the P2X7 cation channel itself; P2X7-stimulated shedding of the homing receptor CD62L; and reversal of activity of the multidrug transporter P-glycoprotein. The data identify a role for PS distribution changes in signal transduction, rapidly modulating the activities of several membrane proteins. This seems to be an all-or-none effect, coordinating the activity of most or all the molecules of a target protein in each cell. The data also suggest a new approach to circumventing multidrug resistance.

The contributions of T-cell anergy to peripheral T-cell tolerance

Intrathymic deletion of thymocytes with high affinity for self antigen cells plays a crucial role in contracting the autoreactive T-cell repertoire. However, this is manifestly an incomplete process. Not all self proteins are effectively presented in the thymus, including those that are expressed well after the bulk of the T-cell repertoire has been formed, and it is relatively easy to detect autoreactive T cells following immunization with self antigens. For this reason mechanisms of regulating peripheral T cells with unwanted specificity are crucial to survival. There are several mechanisms of peripheral T-cell unresponsiveness including ignorance, deletion by apoptosis, and cytokine-mediated regulation. The topic of this review is a further mechanism, T-cell anergy. Data will be highlighted, which suggests that the induction of T-cell anergy is an important contributor to peripheral T-cell tolerance, and that anergic T-cells are not passive, but may play an important role as regulatory cells.

Mechanisms of chemokine and antigen-dependent T-lymphocyte navigation

T-lymphocyte trafficking is targeted to specific organs by selective molecular interactions depending on their differentiation and functional properties. Specific chemokine receptors have been associated with organ-specific trafficking of memory and effector T-cells, as well as the recirculation of naïve T-cells to secondary lymphoid organs. In addition to the acquisition of tissue-selective integrins and chemokine receptors, an additional level of specificity for T-cell trafficking into the tissue is provided by specific recognition of antigen displayed by the endothelium involving the TCRs (T-cell antigen receptors) and co-stimulatory receptors. Activation of PI3K (phosphoinositide 3-kinase) is a robust signalling event shared by most chemokine receptors as well as the TCR and co-stimulatory receptors, contributing to several aspects of T-lymphocyte homing as well as actin reorganization and other components of the general migratory machinery. Accordingly, inhibition of PI3K has been considered seriously as a potential therapeutic strategy by which to combat various T-lymphocyte-dependent pathologies, including autoimmune and inflammatory diseases, as well as to prevent transplant rejection. However, there is substantial evidence for PI3K-independent mechanisms that facilitate T-lymphocyte migration. In this regard, several other signalling-pathway components, including small GTPases, PLC (phospholipase C) and PKC (protein kinase C) isoforms, have also been implicated in T-lymphocyte migration in response to chemokine stimulation. The present review will therefore examine the PI3K-dependent and -independent signal-transduction pathways involved in T-cell migration during distinct modes of T-cell trafficking in response to either chemokines or the TCR and co-stimulatory molecules.

Lactate Regulates Metabolic and Pro-inflammatory Circuits in Control of T Cell Migration and Effector Functions.

Lactate has long been considered a “waste” by-product of cell metabolism, and it accumulates at sites of inflammation. Recent findings have identified lactate as an active metabolite in cell signalling, although its effects on immune cells during inflammation are largely unexplored. Here we ask whether lactate is responsible for T cells remaining entrapped in inflammatory sites, where they perpetuate the chronic inflammatory process. We show that lactate accumulates in the synovia of rheumatoid arthritis patients. Extracellular sodium lactate and lactic acid inhibit the motility of CD4+ and CD8+ T cells, respectively. This selective control of T cell motility is mediated via subtype-specific transporters (Slc5a12 and Slc16a1) that we find selectively expressed by CD4+ and CD8+ subsets, respectively. We further show both in vitro and in vivo that the sodium lactate-mediated inhibition of CD4+ T cell motility is due to an interference with glycolysis activated upon engagement of the chemokine receptor CXCR3 with the chemokine CXCL10. In contrast, we find the lactic acid effect on CD8+ T cell motility to be independent of glycolysis control. In CD4+ T helper cells, sodium lactate also induces a switch towards the Th17 subset that produces large amounts of the proinflammatory cytokine IL-17, whereas in CD8+ T cells, lactic acid causes the loss of their cytolytic function. We further show that the expression of lactate transporters correlates with the clinical T cell score in the synovia of rheumatoid arthritis patients. Finally, pharmacological or antibody-mediated blockade of subtype-specific lactate transporters on T cells results in their release from the inflammatory site in an in vivo model of peritonitis. By establishing a novel role of lactate in control of proinflammatory T cell motility and effector functions, our findings provide a potential molecular mechanism for T cell entrapment and functional changes in inflammatory sites that drive chronic inflammation and offer targeted therapeutic interventions for the treatment of chronic inflammatory disorders.

Mesenchymal stem cells for graft-versus-host disease: close encounters with T cells

Mesenchymal stem cells (MSC) have recently received centre stage attention because of their potent immunosuppressive effect which has also been successfully exploited in the clinical setting to treat graft‐versus‐host disease (GVHD); however, the path to clinical efficacy is hindered by the limited understanding of how MSC work and how best to use their potential. In this issue of the European Journal of Immunology, it is shown, using an animal model, that MSC can treat GVHD only if administered in the presence of active disease and that this requirement is strictly related to the presence of IFN‐γ. Here we summarise the knowledge regarding MSC mediated tolerance and the evidence supporting the notion that MSC must be ‘licensed’ to exert their effects. We also propose the idea that the instrumental effect of IFN‐γ activity on MSC‐mediated immunomodulation relies upon IFN‐γs ability to gather and retain suppressive and effector cells in the same anatomical compartment.

The highway code of T cell trafficking

Coordinated migratory events are required for the development of effective and regulated immunity. Naïve T lymphocytes are programmed to recirculate predominantly in secondary lymphoid tissue by non‐specific stimuli. In contrast, primed T cells must identify specific sites of antigen location in non‐lymphoid tissue to exert targeted effector responses. Following priming, T cells acquire the ability to establish molecular interactions mediated by tissue‐selective integrins and chemokine receptors (homing receptors) that allow their access to specific organs, such as the skin and the gut. Recent studies have shown that an additional level of specificity is provided by the induction of specific T cell migration into the tissue following recognition of antigen displayed by the endothelium. In addition, co‐stimulatory signals (such as those induced by CD28 and CTLA‐4 molecules) have been shown not only to regulate T cell activation and differentiation, but also to orchestrate the anatomy of the ensuing T cell response. Copyright

IFN-alpha subtypes differentially affect human T cell motility.

The type I IFN family includes 14 closely related antiviral cytokines that are produced in response to viral infections. They bind to a common receptor, and have qualitatively similar biological activities. The physiological relevance of this redundancy is still unclear. In this study, we analyzed and compared the effects of two potent antiviral type I IFNs, IFN-α2 and IFN-α8, on the motility of various populations of human T lymphocytes in vitro. In this study, we show that IFN-α2 induces chemokinesis of both CD4+ and CD8+ T cells at various stages of differentiation, and induces functional changes that result in enhanced T cell motility, including up-regulation of the integrins LFA-1 and VLA-4, and subsequently, increased ICAM-1- and fibronectin-dependent migration. In contrast, IFN-α8 did not affect T cell motility, despite having similar antiviral properties and similar effects on the induction of the antiviral protein MxA. However, transcription of other IFN-stimulated genes showed that transcription of these genes is selectively activated by IFN-α2, but not IFN-α8, in T cells. Finally, while the antiviral activity of the two subtypes is inhibited by Abs against the two subunits of the IFN-α receptor, the chemokinetic effect of IFN-α2 is selectively blocked by Abs against the A1 receptor subunit. These observations are consistent with the possibility that subtype-specific intracellular signaling pathways are activated by type I IFNs in T lymphocytes.