CTLA-4 Mediates Inhibitory Function of Mesenchymal Stem/Stromal Cells.

Mesenchymal stem/stromal cells (MSCs) are stem cells of the connective tissue, possess a plastic phenotype, and are able to differentiate into various tissues. Besides their role in tissue regeneration, MSCs perform additional functions as a modulator or inhibitor of immune responses. Due to their pleiotropic function, MSCs have also gained therapeutic importance for the treatment of autoimmune diseases and for improving fracture healing and cartilage regeneration. However, the therapeutic/immunomodulatory mode of action of MSCs is largely unknown. Here, we describe that MSCs express the inhibitory receptor CTLA-4 (cytotoxic T lymphocyte antigen 4). We show that depending on the environmental conditions, MSCs express different isoforms of CTLA-4 with the secreted isoform (sCTLA-4) being the most abundant under hypoxic conditions. Furthermore, we demonstrate that the immunosuppressive function of MSCs is mediated mainly by the secretion of CTLA-4. These findings open new ways for treatment when tissue regeneration/fracture healing is difficult.

A Pronounced Inflammatory Activity Characterizes the Early Fracture Healing Phase in Immunologically Restricted Patients.

Immunologically restricted patients such as those with autoimmune diseases or malignancies often suffer from delayed or insufficient fracture healing. In human fracture hematomas and the surrounding bone marrow obtained from immunologically restricted patients, we analyzed the initial inflammatory phase on cellular and humoral level via flow cytometry and multiplex suspension array. Compared with controls, we demonstrated higher numbers of immune cells like monocytes/macrophages, natural killer T (NKT) cells, and activated T helper cells within the fracture hematomas and/or the surrounding bone marrow. Also, several pro-inflammatory cytokines such as Interleukin (IL)-6 and Tumor necrosis factor α (TNFα), chemokines (e.g., Eotaxin and RANTES), pro-angiogenic factors (e.g., IL-8 and Macrophage migration inhibitory factor: MIF), and regulatory cytokines (e.g., IL-10) were found at higher levels within the fracture hematomas and/or the surrounding bone marrow of immunologically restricted patients when compared to controls. We conclude here that the inflammatory activity on cellular and humoral levels at fracture sites of immunologically restricted patients considerably exceeds that of control patients. The initial inflammatory phase profoundly differs between these patient groups and is probably one of the reasons for prolonged or insufficient fracture healing often occurring within immunologically restricted patients.

Impact of the CTLA-4/CD28 axis on the processes of joint inflammation in rheumatoid arthritis.

OBJECTIVE: The importance of the costimulatory molecules CD28 and CTLA-4 in the pathologic mechanism of rheumatoid arthritis (RA) has been demonstrated by genetic associations and the successful clinical application of CTLA-4Ig for the treatment of RA. This study was undertaken to investigate the role of the CTLA-4/CD28 axis in the local application of CTLA-4Ig in the synovial fluid (SF) of RA patients. METHODS: Quantitative polymerase chain reaction was used to analyze the expression of proinflammatory and antiinflammatory cytokines in ex vivo fluorescence-activated cell sorted CTLA-4+ and CTLA-4- T helper cells from the peripheral blood and SF of RA patients. T helper cells were also analyzed for cytokine expression in vitro after the blockade of CTLA-4 by anti-CTLA-4 Fab fragments or of B7 (CD80/CD86) molecules by CTLA-4Ig. RESULTS: CTLA-4+ T helper cells were unambiguously present in the SF of all RA patients examined, and they expressed increased amounts of interferon-γ (IFNγ), interleukin-17 (IL-17), and IL-10 as compared to CTLA-4- T helper cells. The selective blockade of CTLA-4 in T helper cells from the SF in vitro led to increased levels of IFNγ, IL-2, and IL-17. The concomitant blockade of CD28 and CTLA-4 in T helper cells from RA SF by CTLA-4Ig in vitro resulted in reduced levels of the proinflammatory cytokines IFNγ and IL-2 and increased levels of the antiinflammatory cytokines IL-10 and transforming growth factor ß. CONCLUSION: Our ex vivo and in vitro results demonstrate that the CTLA-4/CD28 axis constitutes a drug target for not only the systemic, but potentially also the local, application of the costimulation blocking agent CTLA-4Ig for the treatment of RA.

Surface CD152 (CTLA-4) expression and signaling dictates longevity of CD28null T cells.

CD28(null) T cells are a highly enriched subset of proinflammatory T cells in patients with autoimmune diseases that are oligoclonal and autoreactive. In this study, we analyzed the role of CD152 signaling on the longevity of human CD28(null) T cells. Using a sensitive staining method for CD152, we show that human CD4(+)CD28(null) and CD8(+)CD28(null) T cells rapidly express surface CD152. Serological inactivation of CD152 using specific Fab or blockade of CD152 ligands using CTLA-4Ig in CD4(+)CD28(null) and CD8(+)CD28(null) T cells enhances apoptosis in a Fas/FasL-dependent manner. CD152 cross-linking on activated CD28(null) cells prevents activation-induced cell death as a result of reduced caspase activity. Apoptosis protection conferred by CD152 is mediated by phosphatidylinositol 3'-kinase-dependent activation of the kinase Akt, resulting in enhanced phosphorylation and thereby inhibition of the proapoptotic molecule Bad. We show that signals triggered by CD152 act directly on activated CD28(null) T lymphocytes and, due to its exclusive expression as a receptor for CD80/CD86 on CD28(null) T cells, prevention of CD152-mediated signaling is likely a target mechanism taking place during therapy with CTLA-4Ig. Our data imply strongly that antagonistic approaches using CD152 signals for chronic immune responses might be beneficial.

Our future in the Anthropocene biosphere.

The COVID-19 pandemic has exposed an interconnected and tightly coupled globalized world in rapid change. This article sets the scientific stage for understanding and responding to such change for global sustainability and resilient societies. We provide a systemic overview of the current situation where people and nature are dynamically intertwined and embedded in the biosphere, placing shocks and extreme events as part of this dynamic; humanity has become the major force in shaping the future of the Earth system as a whole; and the scale and pace of the human dimension have caused climate change, rapid loss of biodiversity, growing inequalities, and loss of resilience to deal with uncertainty and surprise. Taken together, human actions are challenging the biosphere foundation for a prosperous development of civilizations. The Anthropocene reality-of rising system-wide turbulence-calls for transformative change towards sustainable futures. Emerging technologies, social innovations, broader shifts in cultural repertoires, as well as a diverse portfolio of active stewardship of human actions in support of a resilient biosphere are highlighted as essential parts of such transformations.

Integrating the Water Planetary Boundary With Water Management From Local to Global Scales.

The planetary boundaries framework defines the "safe operating space for humanity" represented by nine global processes that can destabilize the Earth System if perturbed. The water planetary boundary attempts to provide a global limit to anthropogenic water cycle modifications, but it has been challenging to translate and apply it to the regional and local scales at which water problems and management typically occur. We develop a cross-scale approach by which the water planetary boundary could guide sustainable water management and governance at subglobal contexts defined by physical features (e.g., watershed or aquifer), political borders (e.g., city, nation, or group of nations), or commercial entities (e.g., corporation, trade group, or financial institution). The application of the water planetary boundary at these subglobal contexts occurs via two approaches: (i) calculating fair shares, in which local water cycle modifications are compared to that context's allocation of the global safe operating space, taking into account biophysical, socioeconomic, and ethical considerations; and (ii) defining a local safe operating space, in which interactions between water stores and Earth System components are used to define local boundaries required for sustaining the local water system in stable conditions, which we demonstrate with a case study of the Cienaga Grande de Santa Marta wetlands in Colombia. By harmonizing these two approaches, the water planetary boundary can ensure that water cycle modifications remain within both local and global boundaries and complement existing water management and governance approaches.

CD25 regulatory T cells determine secondary but not primary remission in EAE: impact on long-term disease progression.

Multiple sclerosis (MS) is often characterized by several relapses and remissions during long-term disease, but neither the responsible cells nor the mechanisms are known to date. Using an animal model of multiple sclerosis, relapsing experimental autoimmune encephalomyelitis (R-EAE) CD4+CD25+ Treg cells expressing Foxp3 and CTLA-4 intracellularly and T lymphocytes expressing surface CTLA-4 were identified in the CNS. The first remission occurred even after depletion of Treg cells, but secondary remissions from EAE were ablated. Despite the unaltered first remission autoantigen rechallenge revealed already an amplified cytokine response during acute phase. These results indicate that the cellular composition during first attack of MS predicts long-term disease progression.

CTLA-4 (CD152) inhibits T cell function by activating the ubiquitin ligase Itch.

CTLA-4 (CD152) is a regulatory molecule in the immune system fundamentally important for the inhibition of T cell activity that is mediated by an unknown mechanism. Here we demonstrate similarities of CTLA-4 and Itch deficient mice and that CTLA-4 deficient T cells show a massive reduction in the overall ubiquitination of proteins. CTLA-4-mediated signal transduction leads to increased de-phosphorylation and therefore activation of the ubiquitin ligase Itch and enhanced ubiquitination of the Itch target molecule JunB. The knock-down of Itch completely abolishes the inhibitory effect of CTLA-4-mediated signal transduction on mRNA accumulation of IFN-gamma and IL-4. These results show that CTLA-4 mediates signals via the activation of the ubiquitin ligase Itch probably leading to the enhanced ubiquitination of Itch target molecules resulting in inhibition of T cell activity.

CTLA-4 (CD152) controls homeostasis and suppressive capacity of regulatory T cells in mice.

OBJECTIVE: CD4+CD25+ regulatory T cells (known as Treg cells) suppress unwanted and autoreactive T cell responses. Treg cells express the costimulatory molecule CTLA-4 intracellularly, but the mechanisms by which Treg cells exploit CTLA-4 signaling remain unclear. The present study was undertaken to investigate the role of CTLA-4 in controlling the homeostasis and suppressive function of Treg cells. METHODS: Murine Treg cells were analyzed by flow cytometry for coexpression of CTLA-4 and typical Treg cell-expressed molecules, and the influence of CTLA-4 on T cell proliferation, suppression, and apoptosis was investigated by in vitro assays. To analyze the importance of CTLA-4 in Treg cell-mediated suppression in vivo, wild-type Treg cells were transferred into CTLA-4-deficient mice displaying lymphoproliferation, and survival was monitored over time. RESULTS: A strong correlation between expression of forkhead box P3 and ex vivo expression of CTLA-4 in Treg cells was observed. Inhibition of CTLA-4 signaling in Treg cells during in vitro stimulation increased cell cycling and led to enhanced activation-induced cell death (AICD), which was mediated by CD95/CD95 ligand-induced activation of caspases. Blockade of CTLA-4 signaling resulted in impairment of the suppressive capacity of Treg cells. Despite these effects, high amounts of Treg cells persisted in CTLA-4-deficient mice. Results of transfer experiments in CTLA-4-deficient mice showed that the mice had a significantly prolonged lifespan when CTLA-4-competent Treg cells were injected. CONCLUSION: Expression of CTLA-4 on Treg cells serves to control T cell proliferation, to confer resistance against AICD, and to maintain the suppressive function of Treg cells.

CD152 (CTLA-4) determines CD4 T cell migration in vitro and in vivo.

BACKGROUND: Migration of antigen-experienced T cells to secondary lymphoid organs and the site of antigenic-challenge is a mandatory prerequisite for the precise functioning of adaptive immune responses. The surface molecule CD152 (CTLA-4) is mostly considered as a negative regulator of T cell activation during immune responses. It is currently unknown whether CD152 can also influence chemokine-driven T cell migration. METHODOLOGY/PRINCIPAL FINDINGS: We analyzed the consequences of CD152 signaling on Th cell migration using chemotaxis assays in vitro and radioactive cell tracking in vivo. We show here that the genetic and serological inactivation of CD152 in Th1 cells reduced migration towards CCL4, CXCL12 and CCL19, but not CXCL9, in a G-protein dependent manner. In addition, retroviral transduction of CD152 cDNA into CD152 negative cells restored Th1 cell migration. Crosslinking of CD152 together with CD3 and CD28 stimulation on activated Th1 cells increased expression of the chemokine receptors CCR5 and CCR7, which in turn enhanced cell migration. Using sensitive liposome technology, we show that mature dendritic cells but not activated B cells were potent at inducing surface CD152 expression and the CD152-mediated migration-enhancing signals. Importantly, migration of CD152 positive Th1 lymphocytes in in vivo experiments increased more than 200% as compared to CD152 negative counterparts showing that indeed CD152 orchestrates specific migration of selected Th1 cells to sites of inflammation and antigenic challenge in vivo. CONCLUSIONS/SIGNIFICANCE: We show here, that CD152 signaling does not just silence cells, but selects individual ones for migration. This novel activity of CD152 adds to the already significant role of CD152 in controlling peripheral immune responses by allowing T cells to localize correctly during infection. It also suggests that interference with CD152 signaling provides a tool for altering the cellular composition at sites of inflammation and antigenic challenge.

The tyrosine phosphatase SHP-2 regulates differentiation and apoptosis of individual primary T lymphocytes.

Although phosphatases are key players of intracellular processes, not much is known about the phosphatase SHP-2 during T cell differentiation. Here we show that ectopic over-expression of SHP-2 in primary T helper cells directly reduced the frequency of individual lymphocytes expressing pro-inflammatory cytokines after antigen-specific stimulation by a mechanism impairing activation of protein kinase C. In addition we demonstrate that SHP-2 mediates enhanced migration upon CXCR4 signaling in a G-protein-dependent manner. Most strikingly, SHP-2 mediated a dramatic increase in apoptosis by highly enhanced activation of caspases. Co-immunoprecipitations of SHP-2 and c-Cbl from primary T helper cells demonstrated that SHP-2 strongly interacts with the ubiquitin ligase c-Cbl, indicating that c-Cbl could mediate the negative signals of SHP-2. Our results show that SHP-2 signal transduction regulates central checkpoints of T cell differentiation by the activation of distinct signaling cascades.

A new role of CTLA-4 on B cells in thymus-dependent immune responses in vivo.

The expression of CTLA-4 (CD152) on the cell surface of B cells and its consequences for the humoral immune response in vivo are unknown. We investigated the expression of CTLA-4 mRNA and protein in B cells in T cell-independent or -dependent ways. B cells in the presence of Ag-stimulated Th2 cells expressed mRNA of CTLA-4 and up-regulated intracellular CTLA-4 protein. Using a liposome-enhanced staining technique, we show for the first time, that surface CTLA-4 protein is expressed by 11-15% of B cells in a T cell-dependent culture system. To dissect the role of CTLA-4 on B cells in vivo, we used bone marrow chimeric mice in which only B cells were CTLA-4 deficient. These mice showed that early B cell development and homeostasis is not influenced by CTLA-4 deficiency of B cells. Ag-specific responses after immunization of the chimeric mice revealed elevated levels of IgM Abs in mice deficient for B cell CTLA-4. We propose that CTLA-4 signals on B cells determine the early fate of B cells in thymus-dependent immune responses.

Multiple functions for CD28 and cytotoxic T lymphocyte antigen-4 during different phases of T cell responses: implications for arthritis and autoimmune diseases.

Chronic T cell responses, as they occur in rheumatoid arthritis, are complex and are likely to involve many mechanisms. There is a growing body of evidence that, in concert with the T cell antigen receptor signal, CD28 and cytotoxic T-lymphocyte antigen-4 (CTLA-4; CD152) are the primary regulators of T cell responses. Whereas CD28 primarily activates T cell processes, CTLA-4 inhibits them. The mechanism for this dichotomy is not fully understood, especially as CD28 and CTLA-4 recruit similar signalling molecules. In addition, recent studies demonstrate that CD28 and CTLA-4 have multiple functions during T cell responses. In particular, CTLA-4 exerts independent distinct effects during different phases of T cell responses that could be exploited for the treatment of rheumatoid arthritis.

Diversity of clonal T cell proliferation is mediated by differential expression of CD152 (CTLA-4) on the cell surface of activated individual T lymphocytes.

Inhibitory effects of CD152 (CTLA-4) engagement during T cell activation have been described. To date, such effects could only be correlated to CD152 expression at the population level because expression of CD152 on the cell surface is too low to be assessed by conventional immunofluorescence on the single cell level. In this study, we use magnetofluorescent liposomes for the immunofluorescent detection of surface CD152-expressing CD4(+) T cells and show that, despite the fact that nearly all cells express intracellular CD152, only a fraction of 12% of activated T cells expresses surface CD152 at any given time point. Surface CD152(+) T cells appear with similar kinetics after primary or secondary activation in vitro. However, the frequency of surface CD152(+) T cells 48 h postactivation is 2-fold higher during secondary activation. Surface expression of CD152 is independent of the proliferative history of an activated T cell. Instruction of T cells for surface expression of CD152 rather depends on the time elapsed since the onset of activation, with a maximum at 48 h, and requires less than 12 h of Ag exposure. CD152(-) T cells, when isolated by cell sorting and restimulated, continue to proliferate. CD152 blockade has no effect on their proliferation. Isolated surface CD152(+) T cells do not proliferate upon restimulation unless CD152 is blocked. CD152 thus acts directly and autonomously on individual activated and proliferating T lymphocytes. Due to its heterogeneous expression on the cell surface of activated Th cells, CD152 might diversify the T cell response.