Functional delineation and differentiation dynamics of human CD4+ T cells expressing the FoxP3 transcription factor.

FoxP3 is a key transcription factor for the development and function of natural CD4(+) regulatory T cells (Treg cells). Here we show that human FoxP3(+)CD4(+) T cells were composed of three phenotypically and functionally distinct subpopulations: CD45RA(+)FoxP3(lo) resting Treg cells (rTreg cells) and CD45RA(-)FoxP3(hi) activated Treg cells (aTreg cells), both of which were suppressive in vitro, and cytokine-secreting CD45RA(-)FoxP3(lo) nonsuppressive T cells. The proportion of the three subpopulations differed between cord blood, aged individuals, and patients with immunological diseases. Terminally differentiated aTreg cells rapidly died whereas rTreg cells proliferated and converted into aTreg cells in vitro and in vivo. This was shown by the transfer of rTreg cells into NOD-scid-common gamma-chain-deficient mice and by TCR sequence-based T cell clonotype tracing in peripheral blood in a normal individual. Taken together, the dissection of FoxP3(+) cells into subsets enables one to analyze Treg cell differentiation dynamics and interactions in normal and disease states, and to control immune responses through manipulating particular FoxP3(+) subpopulations.

Human endothelial cells generate Th17 and regulatory T cells under inflammatory conditions.

Organ transplantation represents a unique therapeutic option for irreparable organ dysfunction and rejection of transplants results from a breakdown in operational tolerance. Although endothelial cells (ECs) are the first target in graft rejection following kidney transplantation, their capacity to alloactivate and generate particular T lymphocyte subsets that could intervene in this process remains unknown. By using an experimental model of microvascular endothelium, we demonstrate that, under inflammatory conditions, human ECs induced proliferation of memory CD4(+)CD45RA(-) T cells and selectively amplified proinflammatory Th17 and suppressive CD45RA(-)HLA-DR(+)FoxP3(bright) regulatory CD4(+) T lymphocytes (Tregs). Although HLA-DR expression on resting microvascular ECs was sufficient to induce proliferation of memory CD4(+) T cells, Treg amplification was dependent on the interaction with CD54, highly expressed only under inflammatory conditions. Moreover, expansion of Th17 cells was dependent on IL-6 and STAT-3, and inhibition of either specifically impaired Th17, without altering Treg expansion. Collectively these data reveal that the HLA-DR(+) ECs regulate the local inflammatory allogeneic response, promoting either an IL-6/STAT-3-dependent Th17 response or a contact-CD54-dependent regulatory response according to the cytokine environment. Finally, these data open therapeutic perspectives in human organ transplantation based on targeting the IL-6/STAT-3 pathway and/or promoting CD54 dependent Treg proliferation.

FoxP3+ regulatory T cells suppress early stages of granuloma formation but have little impact on sarcoidosis lesions.

Sarcoidosis is characterized by a disproportionate Th1 granulomatous immune response in involved organs. It is also associated with both peripheral and intratissular regulatory T cell (Treg) expansion. These cells exhibit powerful antiproliferative activity, yet do not completely inhibit the production of either tumor necrosis factor-alpha or interferon-gamma. The origin of the observed Treg amplification and, more importantly, its impact on the evolution of sarcoidosis remain unresolved issues. Here, we show that CD4(+)CD45RA(-)FoxP3(bright) Tregs proliferate and accumulate within granulomas. However, circulating and tissue Treg numbers are neither correlated with the dissemination of the disease nor correlated locally with the extent of granulomatous inflammation. Rather, we found a positive correlation between the presence of Tregs in renal granulomas and the degree of interstitial fibrosis (r = 0.46, P = 0.03, n = 20). Furthermore, Treg depletion accelerates in vitro granuloma growth in mononuclear cell cultures of healthy controls, but not in those from patients with active sarcoidosis. The results of this study show that although healthy Tregs suppress the initial steps of granuloma formation, they have no positive influence on sarcoidosis lesions. Our findings argue for a more preventive than curative effect of Tregs on inflammatory processes.

Endothelial Cell Amplification of Regulatory T Cells Is Differentially Modified by Immunosuppressors and Intravenous Immunoglobulin.

Immunosuppressive treatment is a prerequisite for both organ transplantation and tolerance of the allograft. However, long-term immunosuppression has been associated with a higher incidence of malignancies and infections. Immunosuppressors mainly target circulating immune cells and little is known of their "off-target" effects, such as their impact on endothelial cells (ECs). In chronic antibody-mediated rejection (AMR), the allograft endothelium is a target of damage, histologically detected as transplant glomerulopathy, and which correlates with poor graft survival. Under inflammatory conditions, EC expression of HLA class II antigens can lead to CD4+-T lymphocyte alloactivation and selective expansion of pro-inflammatory Th17 and pro-tolerance Treg subsets. This response can be modified and preactivation of the EC by HLA-DR antibody binding promoted a proinflammatory Th17 response. However, whether or not immunosuppressors alter EC immunogenicity has not been examined. In alloimmunized patients with AMR, cyclosporine A (CsA) and mycophenolic acid (MPA) are often combined with intravenous immunoglobulins (IVIgs). This study reports changes in the microvascular EC phenotype and function after treatment with CsA, MPA, or IVIg. Both CsA and MPA decreased HLA-DR and increased CD54 expression, whereas IVIg increased HLA-DR expression. Interleukin 6 secretion was reduced by all three immunomodulators. Preincubation of ECs with CsA or MPA limited, while IVIg amplified, Treg expansion. Because CsA, MPA, and IVIg are known for their ability to act upon leukocytes, we confirmed that ECs maintained their immunoregulatory role when allogeneic leukocytes were pretreated with CsA, MPA, or IVIg. The results reveal that individual immunosuppressors, used in the induction and maintenance of renal allograft tolerance, had direct and distinct effects on ECs. Results of experiments associating IVIg with either CsA or MPA underlined the differences observed using individual immunosuppressors. Paradoxically, CsA or MPA may increase EC mediated inflammatory responses and long-term exposure may contribute to limitation of allograft tolerance. In contrast, IVIg interaction with the endothelium may mediate some of its immunosuppressive effects through promotion of Treg expansion, contributing to the maintenance of allograft tolerance.

Study of the allogeneic response induced by endothelial cells expressing HLA class II after lentiviral transduction.

Rejection is still the major cause of allograft loss following organ transplantation and a more complete comprehension of the alloimmune response is required in order to develop new therapeutic approaches. Allogenicity is primarily generated by the expression of major histocompatibility complex (MHC) molecules in the donor organ. Graft microvascular endothelial cells express both HLA class I and class II molecules. They are the first target of the allogeneic response because of their vascular localization and are also able to present antigen to recipient T cells. The endothelium can therefore be considered as both a stimulator of and a target for alloimmune responses and both aspects require further study. We have established a model of constitutive expression of HLA-DR by human microvascular endothelial cells following transduction with a lentiviral vector. This model was employed in a study demonstrating that endothelial cells can induce allogeneic expansion of regulatory and pro-inflammatory CD4(+) T lymphocyte subsets. Because microvascular endothelial cells rapidly lose their expression of HLA-DR ex vivo, this experimental system of lentiviral-mediated expression of HLA-DR allows the study of alloantigen presentation without requiring addition of inflammatory cytokines and thus provides a model for the study of the intra-graft allogeneic CD4(+) T cell response at the single-cell level.

Regulation of the CD4+ T cell allo-immune response by endothelial cells.

Recent studies have revealed the presence of pro-inflammatory and/or regulatory CD4(+) T cells within allografts promoting either graft rejection or tolerance. Histological evidence has identified the microvascular endothelium as the primary site of allograft damage as it is the first site of encounter with the recipient's immune system. The initial view of the human endothelial cell inducing an effector Th1 response leading to graft rejection has been extended by recent results which demonstrate the endothelial cell ability to generate other CD4(+) T cell sub-populations including Th17 and Treg cells. In the transplantation setting, the allo-reactivity of the endothelium with the CD4(+) T cell populations is likely to depend upon multiple factors including the vascular origin of the endothelial cell, the cytokine environment, the presence or absence of pro-inflammatory stimuli including ligands of Toll like receptors and alloantibodies. This review provides an update on the characteristics of the endothelial cell activation of the CD4(+) T cell response and an analysis of the factors, which can modify this activity in favor of either graft rejection or tolerance.

Immunological function of the endothelial cell within the setting of organ transplantation.

In organ transplantation, development of immunosuppressive treatment and improved diagnosis of allograft rejection has resulted in increased allograft survival in recent years. Nevertheless, rejection remains a major cause of graft loss and a better understanding of the characteristics of the allo-immune response is required to identify new diagnostic and therapeutic tools. The allogeneic immune response depends upon a major family of antigenic targets: the Major Histocompatibility Complex molecules (MHC) which are present on donor cells. These molecules are targets of both the humoral and cellular arms of the graft recipient's immune system: T lymphocytes which are implicated in acute cellular rejection and antibodies which are implicated in antibody-mediated rejection (AMR). Allo-recognition of allograft MHC antigens by either T cells or allo-antibodies is the primary event which can ultimately lead to graft rejection. Although immunosuppressive strategies have mainly focused on the T cell response and acute cellular rejection has therefore become relatively rare, antibody mediated rejection (AMR) remains resistant to conventional immunosuppressive treatment and results in frequent graft loss. Damage to the endothelium is a prominent histological feature of AMR underlining the involvement of endothelial cells in initiating the allo-immune response. Furthermore, endothelial cells express both HLA class I and class II molecules in the context of organ transplantation endowing them with the capacity to present antigen to the recipient T cells. The endothelium should therefore be viewed both as a stimulator of, and as a target for allo-immune responses. In this review, we will summarize current knowledge about the implication of endothelial cells in the allo-immune response in the context of organ transplantation.

Regulatory T cells in kidney allograft infiltrates correlate with initial inflammation and graft function.

BACKGROUND: The relevance of borderline change (BL) and subclinical cellular rejection (SCR) observed in renal transplantation remains to be determined. Several studies have shown in BL and SCR the presence of a Th1 immune response, qualitatively similar to but quantitatively reduced in comparison with infiltrates typical of acute cellular rejection (ACR). METHODS: To elucidate the role of regulatory T cells (Tregs) in the local control of the allogenic response, we studied their presence by immunohistochemistry in 24 biopsies with graft dysfunction (12 ACR and 12 BL) and in 16 protocol biopsies at 1 year (eight SCR and eight subclinical BL). RESULTS: The proportion of Tregs in CD4 T infiltrates was higher in BL and SCR when compared with ACR. Moreover, their presence was correlated with the intensity of interstitial inflammation (r=-0.35, P=0.027, n=40) and with graft function at the time of the biopsy (r=-0.37, P=0.018, n=40). CONCLUSION: These data suggest Treg recruitment at the acute phase of the allogenic response, where they could act to diminish the interstitial inflammation and its associated lesions.