Regulation of T‐lymphocyte motility,adhesion and de‐adhesion by a cell surface mechanism directed by low density lipoprotein receptor‐related protein 1 and endogenous thrombospondin‐1

T lymphocytes are highly motile and constantly reposition themselves between a free‐floating vascular state, transient adhesion and migration in tissues. The regulation behind this unique dynamic behaviour remains unclear. Here we show that T cells have a cell surface mechanism for integrated regulation of motility and adhesion and that integrin ligands and CXCL12/SDF‐1 influence motility and adhesion through this mechanism. Targeting cell surface‐expressed low‐density lipoprotein receptor‐related protein 1 (LRP1) with an antibody, or blocking transport of LRP1 to the cell surface, perturbed the cell surface distribution of endogenous thrombospondin‐1 (TSP‐1) while inhibiting motility and potentiating cytoplasmic spreading on intercellular adhesion molecule 1 (ICAM‐1) and fibronectin. Integrin ligands and CXCL12 stimulated motility and enhanced cell surface expression of LRP1, intact TSP‐1 and a 130 000 MW TSP‐1 fragment while preventing formation of a de‐adhesion‐coupled 110 000 MW TSP‐1 fragment. The appearance of the 130 000 MW TSP‐1 fragment was inhibited by the antibody that targeted LRP1 expression, inhibited motility and enhanced spreading. The TSP‐1 binding site in the LRP1‐associated protein, calreticulin, stimulated adhesion to ICAM‐1 through intact TSP‐1 and CD47. Shear flow enhanced cell surface expression of intact TSP‐1. Hence, chemokines and integrin ligands up‐regulate a dominant motogenic pathway through LRP1 and TSP‐1 cleavage and activate an associated adhesion pathway through the LRP1–calreticulin complex, intact TSP‐1 and CD47. This regulation of T‐cell motility and adhesion makes pro‐adhesive stimuli favour motile responses, which may explain why T cells prioritize movement before permanent adhesion.     

T‐cell regulation through a basic suppressive mechanism targeting low‐density lipoprotein receptor‐related protein 1

Cell adhesion is generally considered to depend on positive regulation through ligation of integrins and cytokine receptors. However, here we show that T‐cell adhesion, and notably also T‐cell receptor (TCR) ‐induced activation, are subject to constant suppression through shedding of low‐density lipoprotein receptor‐related protein 1 (LRP1). The broad‐spectrum metalloprotease inhibitor GM6001 abrogated shedding, so inducing prominent cell surface expression of LRP1 while enhancing TCR‐induced activation and adhesion to β₁ and β₂ integrin ligands, hence arresting the cells. Integrin ligands also inhibited shedding but the effect was less potent than that of GM6001. Unlike GM6001, integrin ligands also induced cell surface expression of full‐length thrombospondin‐1 (TSP170) and TSP130, which associated with LRP1, and TSP110, which did not associate with LRP1. Cell surface expression of LRP1 and TSP130 were induced exclusively in adhering cells, expression of TSP110 preferentially in non‐adhering cells and expression of TSP170 correlated with T‐cell motility. The pro‐adhesive chemokine CXCL12 also inhibited LRP1 shedding and induced surface expression of TSP170 and TSP130 while inhibiting TSP110. Exogenous TSP‐1 and ligation of CD28 inhibited shedding although less effectively than GM6001, and the inhibition through CD28 was independent of TSP‐1. Small interfering RNA silencing experiments confirmed involvement of LRP1 and TSP‐1 in integrin‐dependent adhesion and TCR‐induced activation. Hence, the poor LRP1 expression in T cells depends on shedding. Integrin ligands and CXCL12 antagonize shedding through a TSP‐1‐dependent pathway and ligation of CD28 antagonizes shedding independent of TSP‐1. The disappearance of LRP1 from the cell surface may provide basic immunosuppression at the T‐cell level.     

Methotrexate and its therapeutic antagonists caffeine and theophylline,target a motogenic T‐cell mechanism driven by thrombospondin‐1 (TSP‐1)

Methotrexate (MTX) is a widely used treatment for inflammatory diseases such as rheumatoid arthritis and psoriasis, based on the concept that it is immunosuppressive. Its mechanism of action, however, remains unclear, although it is thought to depend on adenosine. Caffeine and theophylline, which have several targets including adenosine receptors, have been shown to suppress the beneficial clinical effects of MTX. Here we show that MTX and caffeine and theophylline differentially affect a motogenic T‐cell mechanism driven by endogenous thrombospondin‐1 (TSP‐1) and its receptor, low density lipoprotein receptor‐related protein 1 (LRP1). MTX stimulated TSP‐1 expression and the motogenic TSP‐1/TSP‐1 receptor mechanism in primary human T cells, hence mimicking IL‐2 and CXCL12, which similar to MTX, dampen inflammatory disease. SiRNA‐mediated gene silencing of TSP‐1 and LRP1 inhibited this stimulatory effect. Caffeine and theophylline inhibited the TSP‐1/TSP‐1 receptor mechanism by inhibiting LRP1 expression. These results indicate that the effect of MTX on T cells is immunoregulatory rather than immunosuppressive, and suggest a pathway dependent on TSP‐1/TSP‐1 receptor interactions for the regulation of immune responses.     

In CD28‐costimulated human naïve CD4+ T cells,I‐κB kinase controls the expression of cell cycle regulatory proteins via interleukin‐2‐independent mechanisms

Stimulation of naïve CD4⁺ T cells through engagement of the T‐cell receptor (TCR) and the CD28 co‐receptor initiates cell proliferation which critically depends on interleukin (IL)‐2 secretion and subsequent autocrine signalling via the IL‐2 receptor. However, several studies indicate that in CD28‐costimulated T cells additional IL‐2‐independent signals are also required for cell proliferation. In this study, using a neutralizing anti‐human IL‐2 antibody and two selective, structurally unrelated, cell‐permeable I‐κB kinase (IKK) inhibitors, BMS‐345541 and PS‐1145, we show that in human naïve CD4⁺ T cells stimulated through a short engagement of the TCR and the CD28 co‐receptor, IKK controls the expression of the cell cycle regulatory proteins cyclin D3, cyclin E and cyclin‐dependent kinase 2 (CDK2) and the stability of the F‐box protein S‐phase kinase‐associated protein 2 (SKP2) and its co‐factor CDC28 protein kinase regulatory subunit 1B (CKS1B), through IL‐2‐independent mechanisms.     

Antigen-induced regulation of T-cell motility,interaction with antigen-presenting cells and activation through endogenous thrombospondin-1 and its receptors

Antigen recognition reduces T-cell motility, and induces prolonged contact with antigen-presenting cells and activation through mechanisms that remain unclear. Here we show that the T-cell receptor (TCR) and CD28 regulate T-cell motility, contact with antigen-presenting cells and activation through endogenous thrombospondin-1 (TSP-1) and its receptors low-density lipoprotein receptor-related protein 1 (LRP1), calreticulin and CD47. Antigen stimulation induced a prominent up-regulation of TSP-1 expression, and transiently increased and subsequently decreased LRP1 expression whereas calreticulin was unaffected. This antigen-induced TSP-1/LRP1 response down-regulated a motogenic mechanism directed by LRP1-mediated processing of TSP-1 in cis within the same plasma membrane while promoting contact with antigen-presenting cells and activation through cis interaction of the C-terminal domain of TSP-1 with CD47 in response to N-terminal TSP-1 triggering by calreticulin. The antigen-induced TSP-1/LRP1 response maintained a reduced but significant motility level in activated cells. Blocking CD28 co-stimulation abrogated LRP1 and TSP-1 expression and motility. TCR/CD3 ligation alone enhanced TSP-1 expression whereas CD28 ligation alone enhanced LRP1 expression. Silencing of TSP-1 inhibited T-cell conjugation to antigen-presenting cells and T helper type 1 (Th1) and Th2 cytokine responses. The Th1 response enhanced motility and increased TSP-1 expression through interleukin-2, whereas the Th2 response weakened motility and reduced LRP1 expression through interleukin-4. Ligation of the TCR and CD28 therefore elicits a TSP-1/LRP1 response that stimulates prolonged contact with antigen-presenting cells and, although down-regulating motility, maintains a significant motility level to allow serial contacts and activation. Th1 and Th2 cytokine responses differentially regulate T-cell expression of TSP-1 and LRP1 and motility.     

Type I interferon potentiates T‐cell receptor mediated induction of IL‐10‐producing CD4+ T cells

Type I interferons (IFNs) have the dual ability to promote the development of the immune response and exert an anti‐inflammatory activity. We analyzed the integrated effect of IFN‐α, TCR signal strength, and CD28 costimulation on human CD4⁺ T‐cell differentiation into cell subsets producing the anti‐ and proinflammatory cytokines IL‐10 and IFN‐γ. We show that IFN‐α boosted TCR‐induced IL‐10 expression in activated peripheral CD45RA⁺CD4⁺ T cells and in whole blood cultures. The functional cooperation between TCR and IFN‐α efficiently occurred at low engagement of receptors. Moreover, IFN‐α rapidly cooperated with anti‐CD3 stimulation alone. IFN‐α, but not IL‐10, drove the early development of type I regulatory T cells that were mostly IL‐10⁺ Foxp3^? IFN‐γ^? and favored IL‐10 expression in a fraction of Foxp3⁺ T cells. Our data support a model in which IFN‐α costimulates TCR toward the production of IL‐10 whose level can be amplified via an autocrine feedback loop.     

Stress‐activated dendritic cells interact with CD4+ T cells to elicit homeostatic memory

Evidence is presented that thermal or oxidizing stress‐activated DC interact with CD4⁺ T cells to induce and maintain a TCR‐independent homeostatic memory circuit. Stress‐activated DC expressed endogenous intra‐cellular and cell surface HSP70. The NF‐κB signalling pathway was activated and led to the expression of membrane‐associated IL‐15 molecules. These interacted with the IL‐15 receptor complex on CD4⁺ T cells, thus activating the Jak3 and STAT5 phosphorylation signalling pathway to induce CD40 ligand expression, T‐cell proliferation and IFN‐γ production. CD40 ligand on CD4⁺ T cells in turn re‐activated CD40 molecules on DC, inducing DC maturation and IL‐15 expression thereby maintaining the feedback circuit. The proliferating CD4⁺ T cells were characterized as CD45RA^? CD62L⁺ central memory cells, which underwent homeostatic proliferation. The circuit is independent of antigen and MHC‐class‐II‐TCR interaction as demonstrated by resistance to TCR inhibition by ZAP70 inhibitor or MHC‐class II antibodies. These findings suggest that stress can activate a DC‐CD4⁺ T‐cell interacting circuit, which may be responsible for maintaining a homeostatic antigen‐independent memory.     

Hypoxia‐inducible factor‐1α perpetuates synovial fibroblast interactions with T cells and B cells in rheumatoid arthritis

Synovial fibroblast hyperplasia, T‐cell hyperactivity, B‐cell overactivation, and the self‐perpetuating interactions among these cell types are major characteristics of rheumatoid arthritis (RA). The inflamed joints of RA patients are hypoxic, with upregulated expression of hypoxia‐inducible factor‐1α (HIF‐1α) in RA synovial fibroblasts (RASFs). It remains unknown whether HIF‐1α regulates interactions between RASFs and T cells and B cells. We report here that HIF‐1α promotes the expression of inflammatory cytokines IL‐6, IL‐8, TNF‐α, and IL‐1β, and cell–cell contact mediators IL‐15, vascular cell adhesion molecule (VCAM)‐1, thrombospondin (TSP)‐1, and stromal cell‐derived factor (SDF)‐1 in RASFs. Furthermore, HIF‐1α perpetuates RASF‐mediated inflammatory Th1‐ and Th17‐cell expansion while differentially inhibiting regulatory B10 and innate‐like B cells, leading to increased IFN‐γ, IL‐17, and IgG production and decreased protective natural IgM secretion. Our findings suggest that HIF‐1α perpetuates the interactions between RASFs and T cells and B cells to induce inflammatory cytokine and autoantibody production, thus exacerbating the severity of RA. Targeting HIF‐1α may provide new therapeutic strategies for overcoming this persistent disease.     

Arginase‐1 is neither constitutively expressed in nor required for myeloid‐derived suppressor cell‐mediated inhibition of T‐cell proliferation

Although previous reports suggest that tumor‐induced myeloid‐derived suppressor cells (MDSC) inhibit T cells by L‐arginine depletion through arginase‐1 activity, we herein show that arginase‐1 is neither inherently expressed in MDSC nor required for MDSC‐mediated inhibition. Employing Percoll density gradients, large expansions of MDSC in the bone marrow of tumor‐bearing mice were isolated and demonstrated potent inhibition in T‐cell proliferation activated by TCR‐ligation, Concanavalin A, PMA plus ionomycin, or IL‐2. Despite demonstrating characteristic immunosuppressive capacity, these MDSC exhibit no arginase‐1 expression and/or exert their inhibitory effects independent of arginase‐1 activity. However, arginase‐1 expression in MDSC can be induced by exposure to TCR‐activated T cells or their culture medium, but not T cells activated by other means or growing tumor cells. Further investigation reveals multiple cytokines secreted by TCR‐activated T cells as orchestrating two signaling‐relay axes, IL‐6‐to‐IL‐4 and GM‐CSF/IL‐4‐to‐IL‐10, leading to arginase‐1 expression in MDSC. Specifically, IL‐6 signaling increases IL‐4R, enabling IL‐4 to induce arginase‐1 expression; similarly, GM‐CSF in concert with IL‐4 induces IL‐10R, allowing IL‐10‐mediated induction. Surprisingly, our study indicates that induction of arginase‐1 expression is not conducive to the critical MDSC‐mediated inhibition toward T cells, which is rather dependent on direct cell contacts undiminished by PD‐L1 blockade or SIRPα deficiency.     

A novel Zap70 mutation with reduced protein stability demonstrates the rate‐limiting threshold for Zap70 in T‐cell receptor signalling

Loss of ζ‐associated protein 70 (Zap70) results in severe immunodeficiency in humans and mice because of the critical role of Zap70 in T‐cell receptor (TCR) signalling. Here we describe a novel mouse strain generated by N‐ethyl‐N‐nitrosourea mutagenesis, with the reduced protein stability (rps) mutation in Zap70. The A243V rps mutation resulted in decreased Zap70 protein and a reduced duration of TCR‐induced calcium responses, equivalent to that induced by a 50% decrease in catalytically active Zap70. The reduction of signalling through Zap70 was insufficient to substantially perturb thymic differentiation of conventional CD4 and CD8 T cells, although Foxp3⁺ regulatory T cells demonstrated altered thymic production and peripheral homeostasis. Despite the mild phenotype, the Zap70^A243V variant lies just above the functional threshold for TCR signalling competence, as T cells relying on only a single copy of the Zap70^rps allele for TCR signalling demonstrated no intracellular calcium response to TCR stimulation. This addition to the Zap70 allelic series indicates that a rate‐limiting threshold for Zap70 protein levels exists at which signalling capacity switches from nearly intact to effectively null.     

IL‐15 dependent induction of IL‐18 secretion as a feedback mechanism controlling human MAIT‐cell effector functions

Mucosal‐associated invariant T (MAIT) cells are characterized by an invariant TCRVα7.2 chain recognizing microbial vitamin B metabolites presented by the MHC‐Ib molecule MR1. They are mainly detectable in the CD8⁺ and CD8^?CD4^? “double negative” T‐cell compartments of mammals and exhibit both Th1‐ and Th17‐associated features. As MAIT cells show a tissue‐homing phenotype and operate at mucosal surfaces with myriads of pathogenic encounters, we wondered how IL‐15, a multifaceted cytokine being part of the intestinal mucosal barrier, impacts on their functions. We demonstrate that in the absence of TCR cross‐linking, human MAIT cells secrete IFN‐γ, increase perforin expression and switch on granzyme B production in response to IL‐15. As this mechanism was dependent on the presence of CD14⁺ cells and sensitive to IL‐18 blockade, we identified IL‐15 induced IL‐18 production by monocytes as an inflammatory, STAT5‐dependent feedback mechanism predominantly activating the MAIT‐cell population. IL‐15 equally affects TCR‐mediated MAIT‐cell functions since it dramatically amplifies bacteria‐induced IFN‐γ secretion, granzyme production, and cytolytic activity at early time points, an effect being most pronounced under suboptimal TCR stimulation conditions. Our data reveal a new quality of IL‐15 as player in an inflammatory cytokine network impacting on multiple MAIT‐cell functions.     

IL‐15 regulates Bcl‐2 family members Bim and Mcl‐1 through JAK/STAT and PI3K/AKT pathways in T cells

Maintenance of T cells is determined by their survival capacity, which is regulated by Bcl‐2 proteins. Cytokines signalling through the common gamma chains such as IL‐2, IL‐7 and IL‐15 are important for T‐cell survival but how these cytokines determine the expression of Bcl‐2‐family proteins is not clear. We report signalling events of cytokines that regulate expression of two key Bcl‐2 proteins, pro‐apoptotic Bim and anti‐apoptotic Mcl‐1, in resting C57BL/6 mouse T cells. IL‐2, IL‐7 and IL‐15 inhibited apoptosis but paradoxically induced the expression of Bim, countered by concomitant induction of Mcl‐1. Bim induction by IL‐15 was found at the mRNA and protein levels and depended on both JAK/STAT and PI3K signals. A new STAT5‐binding site was identified in the Bim promoter, which was occupied by STAT5 upon IL‐15 stimulation. Although it also depended on JAK/STAT‐ and PI3K signalling, Mcl‐1 regulation was independent of Mcl‐1 mRNA levels and of regulation of protein stability, suggesting translational regulation. Concurrent CD3 signals inhibited some of the IL‐7 effect but not the IL‐15 effect on Bcl‐2 proteins. The data suggest that cytokines induce Bim and prime T cells for apoptosis, but also inhibit apoptosis by stabilising Mcl‐1. Later downregulation of short‐lived Mcl‐1 may induce efficient, Bim‐dependent apoptosis.     

Interleukin‐10‐secreting T cells define a suppressive subset within the HIV‐1‐specific T‐cell population

Recent studies have indicated that Treg contribute to the HIV type 1 (HIV‐1)‐related immune pathogenesis. However, it is not clear whether T cells with suppressive properties reside within the HIV‐1‐specific T‐cell population. Here, PBMC from HIV‐1‐infected individuals were stimulated with a 15‐mer Gag peptide pool, and HIV‐1‐specific T cells were enriched by virtue of their secretion of IL‐10 or IFN‐γ using immunomagnetic cell‐sorting. Neither the IL‐10‐secreting cells nor the IFN‐γ‐secreting cells expressed the Treg marker FOXP3, yet the IL‐10‐secreting cells potently suppressed anti‐CD3/CD28‐induced CD4⁺ as well as CD8⁺ T‐cell proliferative responses. As shown by intracellular cytokine staining, IL‐10‐ and IFN‐γ‐producing T cells represent distinct subsets of the HIV‐1‐specific T cells. Our data collectively suggest that functionally defined HIV‐1‐specific T‐cell subsets harbor potent immunoregulatory properties that may contribute to HIV‐1‐associated T‐cell dysfunction.     

Cell contact interaction between adipose‐derived stromal cells and allo‐activated T lymphocytes

Mesenchymal stromal cells regulate immune cell function via the secretion of soluble factors. Cell membrane interactions between these cell types may play an additional role. Here, we demonstrate that subpopulations of allo‐activated T cells are capable of binding to human adipose‐derived stromal cells (ASC). The bound T‐cell population contained CD8⁺ T cells and was enriched for CD4^?CD8^? T cells, whereas the proportion of CD4⁺ T cells was decreased compared with the non‐bound T‐cell population. Bound CD4⁺ T cells had high proliferative activity and increased CD25 and FoxP3 expression. However, they also expressed CD127, excluding regulatory T‐cell function. In CD8⁺ T cells, IL‐2 sensitivity, as determined by the analysis of phosphorylated STAT5, was lower in the presence of ASC and even lower in bound cells. In contrast, IL‐2‐induced phosphorylated STAT5 levels were higher in bound CD4⁺ T cells than in non‐bound CD4⁺ T cells. Additionally, pro‐proliferative TGF‐β signalling via endoglin and SMAD1/5/8 phosphorylation was detected in bound CD4⁺ T cells. Even after prolonged co‐culture with ASC, the activated phenotype of bound CD4⁺ T cells persisted. In conclusion, these results demonstrate that the binding of lymphocytes to ASC represents an immunomodulatory mechanism in which CD8⁺ T cells are inhibited in their responsiveness to pro‐inflammatory stimuli and reactive CD4⁺ T cells are depleted from the immune response.     

IL‐4‐induced gene 1 maintains high Tob1 expression that contributes to TCR unresponsiveness in human T helper 17 cells

Human Th17 cells have a limited proliferative capacity compared to other T‐cell subsets. We have shown that human Th17 cells display impaired IL‐2 production due to IL‐4‐induced gene 1 (IL4I1) upregulation. Here, we show that in human Th17 cells, IL4I1 also maintains high levels of Tob1, a member of the Tob/BTG (B‐cell traslocation gene) antiproliferative protein family, which prevents cell‐cycle progression mediated by TCR stimulation. Indeed, Th17 cells exhibited higher levels of Tob1 than Th1 cells in both resting and TCR‐activated conditions. Accordingly, the expression of positive regulators of the cell cycle (cyclin A, B, C, and E and Cdk2), as well as of Skp2, which promotes Tob1 degradation, was lower in Th17 cells than in Th1 cells. Tob1 expression in human Th17 cells correlated with both RAR (retinoic acid receptor)‐related orphan receptor C (RORC) and IL4I1 levels. However, RORC was not directly involved in the regulation of Tob1 expression, whereas IL4I1 silencing in Th17 cells induced a substantial decrease of Tob1 expression. These data suggest that IL4I1 upregulation in human Th17 cells limits their TCR‐mediated expansion not only by blocking the molecular pathway involved in the activation of the IL‐2 promoter, but also by maintaining high levels of Tob1, which impairs entry into the cell cycle.     

The microRNA‐9/B‐lymphocyte‐induced maturation protein‐1/IL‐2 axis is differentially regulated in progressive HIV infection

The fine control of T‐cell differentiation and its impact on HIV disease states is poorly understood. In this study, we demonstrate that B‐lymphocyte‐induced maturation protein‐1 (Blimp‐1/Prdm1) is highly expressed in CD4⁺ T cells from chronically HIV‐infected (CHI) patients compared to cells from long‐term nonprogressors or healthy controls. Stimulation through the T‐cell receptor in the presence ofIL‐2 induces Blimp‐1 protein expression. We show here that Blimp‐1 levels are translationally regulated by microRNA‐9 (miR‐9). Overexpression of miR‐9 induces Blimp‐1 repression, restoring IL‐2 secretion in CD4⁺ T cells via reduction in the binding of Blimp‐1 to the il‐2 promoter. In CHI patients where IL‐2 expression is reduced and there is generalized T‐cell dysfunction, we show differential expression of both miR‐9 and Blimp‐1 in CD4⁺ cells compared with levels in long‐term nonprogressors. These data identify a novel miR‐9/Blimp‐1/IL‐2 axis that is dysregulated in progressive HIV infection.