T cell trafficking and metabolism: novel mechanisms and targets for immunomodulation.

Coordinated migratory events by naïve and memory T cells are key to effective immunity. Naïve T cells predominantly recirculate through secondary lymphoid tissue until antigen encounter, while primed T cells efficiently localize to antigen-rich lymphoid and non-lymphoid tissue. Tissue-selective targeting by primed T cells is achieved by a combination of inflammatory signals and tissue-selective homing receptors acquired by T cells during activation and differentiation. A large number of molecular mediators and interactions promoting memory T cell migration to non-lymphoid sites of inflammation have been identified. Recently, additional antigen-driven mechanisms have been proposed, which orchestrate the targeted delivery of memory T cells to antigen-rich tissue. Importantly, recent studies have revealed that the T cell metabolic status influences their differentiation and homing patterns. We here summarize these key observations and discuss their relevance for the manipulation of immune anatomy in therapeutic settings.

Regulation of T-cell migration by co-stimulatory molecules.

Migration of primed T-cells to the antigenic site is an essential event in the development of effective immunity. This process is tightly regulated in order to ensure efficient and specific responses. Most studies have focused on non-specific mediators of T-cell migration, including integrins and chemokines. However, recent studies have highlighted the key role of the T-cell receptor and co-stimulatory molecules in guiding T-cell access to antigenic tissue. Here, we review the experimental evidence for an essential contribution of co-stimulation-mediated molecular interactions regulating T-cell migration in the development of T-cell immunity and tolerance.

A rapid method for labelling CD4+ T cells with ultrasmall paramagnetic iron oxide nanoparticles for magnetic resonance imaging that preserves proliferative, regulatory and migratory behaviour in vitro.

A number of techniques have been developed to track the migration of T cells in vivo, but they all suffer significant shortcomings, including the examination of selected organs rather than the organism as a whole--thus precluding longitudinal studies--or limitations imposed by poor spatial resolution and the application of ionizing radiation. By conjugating the HIV tat peptide to ultrasmall superparamagnetic iron oxide (USPIO) nanoparticles in a reaction yielding a mean valence of 45, a magnetic resonance (MR) contrast agent was synthesised that allowed T cells to be efficiently labelled within just 5 min. The USPIO nanoparticles were incorporated into both the cytoplasm and nucleus of labelled cells, which retained normal in vitro proliferative responses to a polyclonal stimulus; suppressive responses mediated by labelled CD4(+) CD25(+) regulatory T cells; chemotactic responses to the chemokine CXCL-12; and transmigration of an activated endothelial monolayer. We believe that this rapid, efficient and essentially non-toxic approach to labelling both murine and human T cells for MRI holds considerable promise, paving the way for the wider immunological application of this exciting technology.

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.

Activated murine endothelial cells have reduced immunogenicity for CD8+ T cells: a mechanism of immunoregulation?

The immunogenic properties of primary cultures of murine lung microvascular endothelial cells (EC) were analyzed. Resting endothelial cells were found to constitutively express low levels of MHC class I and CD80 molecules. IFN-gamma treatment of EC resulted in a marked up-regulation of MHC class I, but no change was observed in the level of CD80 expression. No CD86 molecules were detectable under either condition. The ability of peptide-pulsed EC to induce the proliferation of either the HY-specific, H2-K(k)-restricted CD8(+) T cell clone (C6) or C6 TCR-transgenic naive CD8(+) T cells was analyzed. Resting T cells were stimulated to divide by quiescent peptide-prepulsed EC, while peptide-pulsed, cytokine-activated EC lost the ability to induce T cell division. Furthermore, Ag presentation by cytokine-activated EC induced CD8(+) T cell hyporesponsiveness. The immunogenicity of activated EC could be restored by adding nonsaturating concentrations of anti-H2-K(k) Ab in the presence of an optimal concentration of cognate peptide. This is consistent with the suggestion that the ratio of TCR engagement to costimulation determines the outcome of T cell recognition. In contrast, activated peptide-pulsed EC were killed more efficiently by fully differentiated effector CD8(+) T cells. Finally, evidence is provided that Ag recognition of EC can profoundly affect the transendothelial migration of CD8(+) T cells. Taken together, these results suggest that EC immunogenicity is regulated in a manner that contributes to peripheral tolerance.

Lack of T cell proliferation without induction of nonresponsiveness after antigen presentation by endothelial cells.

BACKGROUND: After priming or reactivation in lymph nodes, T cells recirculate to sites of inflammation, and enter tissues by migrating across activated endothelium. Given that activated endothelial and tissue parenchymal cells express both class I and class II MHC molecules, it is probable that transmigrating T cells encounter cognate antigen on endothelial cells, and on tissue parenchymal cells once they have entered the tissue. METHODS: In this study the consequences of antigen presentation by endothelial and epithelial cells to human CD4+ T cell clones were analyzed and compared by a two-step culture system. RESULTS: T cell clones that required B7-mediated costimulation to be activated were found not to be able to proliferate to antigen presented by either endothelial or epithelial cells, unless trans-costimulation was provided by the addition of B7-transfected cells in the cultures. Furthermore, antigen presentation by epithelial cells induced nonresponsiveness in the T cell clones. In contrast, after cognate recognition on endothelial cells, the ability of the T cell clones to proliferate to a subsequent rechallenge with antigen presented on a specialized APC was unaffected. CONCLUSIONS: These data suggest that endothelial cells have unique properties as antigen-presenting cells, in that they do not influence the subsequent reactivity of cognate T cells.

Recently activated T cells are costimulation-dependent in vitro.

While the prominent role of B7-mediated signaling in the activation of naive and resting T cells has been exhaustively demonstrated, it is unclear whether costimulation is required in the amplification of an initiated immune response. In this study we have developed a multistep culture system to investigate the costimulation requirements of recently activated alloreactive CD4(+) T cells and the outcome of allorecognition of B7-deficient, MHC class-II-expressing epithelial cells. The results show that following in vitro "priming" with allogeneic costimulation rich antigen presenting cells, T cells can be reactivated to proliferate only if B7-mediated costimulation is provided. Furthermore, recognition of antigen on B7-negative epithelial cells induced allospecific nonresponsiveness in the responder T cells. Finally, the nonresponsive state was not accompanied by IL-4 secretion and appeared to be reversible, since T cell reactivity could be restored by short-term culture in the presence of IL-2. These observations suggest that "primed" T cells remain B7-dependent in vitro and are susceptible to functional inactivation following costimulation-deficient antigen presentation.

Antigen presentation by parenchymal cells: a route to peripheral tolerance?

T-cell activation and the development of efficient immune responses requires the delivery, by the antigen-presenting cell, of two distinct signals. The first results from the engagement of the TCR:CD3:CD4 complex, and the second from the interaction of CD28 with the B7 family of co-stimulatory molecules. In this context, the physiological significance and the functional consequences of antigen presentation by B7-deficient parenchymal cells, which express MHC class II molecules as a result of inflammation, remains a matter of debate. In this paper we have attempted to critically review the often conflicting reports on the functional effects of antigen presentation by epithelial and endothelial cells to T cells, both in vitro and in vivo. Our own findings are summarised in a model which is consistent with the suggestion of an important role for antigen presentation by parenchymal cells in the induction and the maintenance of peripheral tolerance.

Role of donor and recipient antigen-presenting cells in priming and maintaining T cells with indirect allospecificity.

BACKGROUND: It has been suggested that the sensitization of recipient T lymphocytes against peptides derived from allogeneic major histocompatibility complex (MHC) antigens in the context of self-MHC molecules may contribute to the pathogenesis of chronic allograft rejection. The purpose of this study was to quantitate and characterize the indirect alloresponse in renal transplantation. METHODS: An HLA-A2-negative patient whose A2-positive kidney transplant failed as a result of chronic rejection was selected for this study. T-cell clones were raised using a cocktail of peptides corresponding to polymorphic regions of the A2 sequence and studied by measuring their proliferation using [3H]thymidine incorporation. The presence in vivo of HLA-A2-specific T cells was assessed using limiting dilution analysis. RESULTS: T-cell clones were specific for a single peptide of HLA-A2, residues 92-120, and restricted by HLA-DRB1*1502. The frequency of interleukin-2-secreting T cells specific for this A2 peptide was 1:86,000, only 2-fold lower than that measured against the recall antigen tetanus toxoid. Capitalizing on the similarity of the donor and recipient DR15 alleles (DRB1*1501 and 1502), the question was addressed as to how these T cells had been primed in vivo. Although the large majority of clones responded to A2 synthetic peptide presented by both DR15 alleles, only 3 of 10 clones responded to cells co-expressing DRB1*1501 and A2. CONCLUSION: These data suggest that antigen presentation by recipient APCs is responsible for maintaining T cells with indirect allospecificity in vivo and that, in the context of partial DR matching, indirect presentation by the parenchymal cells of the graft may serve to induce tolerance in T cells with indirect allospecificity.

Detection of CD40 on human thyroid follicular cells: analysis of expression and function.

Thyroid follicular cells (TFC) are a common target of autoimmune attack, but the role they play in inciting and maintaining this attack is unclear. TFC express cytokines, adhesion molecules, and class I and II major histocompatibility complex molecules, but without additional signals that costimulate T cells, they may down-regulate, rather than stimulate, T cell function. In this report, we have investigated whether TFC can express the CD40 molecule, which plays a crucial role in the reciprocal two-way communication between T and B cells. We have shown by immunohistochemistry and flow cytometry that CD40 is expressed by TFC in vivo and in vitro in both autoimmune and nonautoimmune glands. CD40 expression was up-regulated by interleukin-1alpha and interferon-gamma, but not by TSH. Although there was no significant effect of CD40 ligation on cAMP synthesis or [3H]thymidine incorporation, there was a significant increase in interleukin-6 release by TFC. Thus, although TFC do not express members of the B7 family of T cell costimulators, they do express CD40, indicating the possibility of mutually stimulatory T cell-TFC interaction. This has important implications, both for TFC synthesis of immunological mediators and for the biasing of T cell behavior toward a T helper 2-type phenotype.

Interferon-gamma-treated renal tubular epithelial cells induce allospecific tolerance.

Following organ transplantation, tissue parenchymal cells commonly express major histocompatibility complex (MHC) class II molecules as a result of local cytokine release, and thus acquire the capacity to present donor MHC alloantigens to alloreactive CD4+ T cells. The consequences of such a presentation are likely to be relevant in the induction of tolerance to the transplanted tissues, and this has been reported in animal models of transplantation and in humans. In this study, the consequences of antigen presentation by interferon-gamma (IFN-gamma)-treated human renal tubular epithelial cells (RTEC) to resting and activated CD4+ T cells were investigated. Allogeneic RTEC were unable to stimulate proliferation by peripheral blood CD45 RA+ or RO+ CD4+ T cells from three HLA-mismatched responders. The response to RTEC was partially reconstituted by the addition of murine L cell transfectants expressing human B7.1 (DAP.3-B7), suggesting that the failure of RTEC to stimulate a primary alloresponse was due, at least in part, to a lack of costimulation. T cell clones dependent on B7-mediated co-stimulation also did not respond to peptide presented by RTEC. Most importantly, this lack of reactivity was accompanied by the induction of nonresponsiveness. Incubation with allogeneic, DR-expressing RTEC induced allospecific hyporesponsiveness in both CD45RA+ and RO+ cells. Similarly, overnight incubation with antigen-pulsed RTEC induced nonresponsiveness in the B7-dependent T cell clones. These results suggest that MHC class II expression on RTEC may contribute to the induction of an allospecific nonresponsiveness following organ transplantation.

Antigen presentation by epithelial cells induces anergic immunoregulatory CD45RO+ T cells and deletion of CD45RA+ T cells.

The immunoregulatory effects of alloantigen presentation by tissue parenchymal cells to resting peripheral blood CD4+ T cells was investigated. Coculture of CD45RO+ (memory) and CD45RA+ (naive) T lymphocytes with primary cultures of MHC class II-expressing epithelial cells rendered both populations of T cells hyporesponsive to a subsequent challenge by the same MHC molecule expressed on EBV-transformed lymphoblastoid B cell lines. However, the mechanisms responsible for the allospecific hyporesponsiveness were distinct. For the CD45RO+ T cells, responsiveness was restored by subsequent culture in the presence of IL-2; the addition of IL-2 had no effect on the reactivity of the CD45RA+ T cells. In contrast, the naive T cells were protected from the induction of nonresponsiveness by the presence of a neutralizing anti-CD95 Ab during the culture with thyroid follicular cells. In addition, the hyporesponsive CD45RO+ T cells effected linked suppression, in that they inhibited proliferation against a third-party DR alloantigen when the third-party alloantigen was coexpressed with the DR Ag against which hyporesponsiveness had been induced. These results suggest that recognition of Ag by T cells on tissue parenchymal cells plays an important role in the maintenance of peripheral T cell tolerance, inducing nonresponsiveness in naive and memory T cells by distinct mechanisms.