Cytotoxic T lymphocyte antigen 4 (CTLA-4) is an essential negative regulator of T cell immune responses whose mechanism of action is the subject of debate. CTLA-4 shares two ligands (CD80 and CD86) ...with a stimulatory receptor, CD28. Here, we show that CTLA-4 can capture its ligands from opposing cells by a process of trans-endocytosis. After removal, these costimulatory ligands are degraded inside CTLA-4—expressing cells, resulting in impaired costimulation via CD28. Acquisition of CD86 from antigen-presenting cells is stimulated by T cell receptor engagement and observed in vitro and in vivo. These data reveal a mechanism of immune regulation in which CTLA-4 acts as an effector molecule to inhibit CD28 costimulation by the cell-extrinsic depletion of ligands, accounting for many of the known features of the CD28—CTLA-4 system.
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BFBNIB, NMLJ, NUK, PNG, SAZU, UL, UM, UPUK
CD28 and CTLA-4 (CD152) play essential roles in regulating T cell immunity, balancing the activation and inhibition of T cell responses, respectively. Although both receptors share the same ligands, ...CD80 and CD86, the specific requirement for two distinct ligands remains obscure. In the present study, we demonstrate that, although CTLA-4 targets both CD80 and CD86 for destruction via transendocytosis, this process results in separate fates for CTLA-4 itself. In the presence of CD80, CTLA-4 remained ligand bound, and was ubiquitylated and trafficked via late endosomes and lysosomes. In contrast, in the presence of CD86, CTLA-4 detached in a pH-dependent manner and recycled back to the cell surface to permit further transendocytosis. Furthermore, we identified clinically relevant mutations that cause autoimmune disease, which selectively disrupted CD86 transendocytosis, by affecting either CTLA-4 recycling or CD86 binding. These observations provide a rationale for two distinct ligands and show that defects in CTLA-4-mediated transendocytosis of CD86 are associated with autoimmunity.
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EMUNI, FIS, FZAB, GEOZS, GIS, IJS, IMTLJ, KILJ, KISLJ, MFDPS, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, SBMB, SBNM, UKNU, UL, UM, UPUK, VKSCE, ZAGLJ
The immune suppressive protein CTLA-4 is constitutively expressed by Tregs and induced in effector T cells upon activation. Its crucial role in adaptive immunity is apparent from the fatal autoimmune ...pathology seen in CTLA-4 knockout mice. However, little is known regarding factors that regulate CTLA-4 expression and their effect upon its function to remove CD80 and CD86 from antigen presenting cells by transendocytosis. Th17 cells are emerging as significant players in autoimmunity as well as other diseases. Therefore, in this study we have examined the effects of Th17 polarising conditions on CTLA-4 expression and function in human T cells and show that Th17 conditions can suppress the expression of CTLA-4 and its transendocytic function. In contrast to Th17 cells, vitamin D is inversely associated with autoimmune disease. We have previously shown a striking ability of 1,25 dihydroxyvitamin D3 (1,25(OH)2D3) to enhance CTLA-4, however, its effects upon B7 transendocytosis and its activity in the context of inflammation remained unknown. Here we show that induction of CTLA-4 by 1,25(OH)2D3 can actually be enhanced in the presence of Th17 polarising cytokines. Furthermore, its transendocytic function was maintained such that T cells generated in the presence of Th17 conditions and 1,25(OH)2D3 were highly effective at capturing CTLA-4 ligands from antigen presenting cells and suppressing T cell division. Taken together, these data reveal an inhibitory effect of Th17 polarising conditions upon CTLA-4-mediated regulation and show that 1,25(OH)2D3 counteracts this effect. Given the importance of CTLA-4-mediated suppression in the control of autoimmune diseases, our novel data highlight the importance of vitamin D in inflammatory settings.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
(ArJ) is a Mediterranean aromatic plant used traditionally to treat gastrointestinal ailments, skin diseases, atherosclerosis, and as an immuno-stimulant. This study describes ArJ essential oil ...constituents and investigates their wound healing activity. The in vitro antioxidant and antibiofilm activities of ArJ essential oil were investigated. The in vivo pro/anti-inflammatory and oxidative/antioxidant markers were compared with standard silver sulfadiazine (SS) in a second-degree skin burn experimental rat model. The gas chromatography-equipped flame ionization detector (GC-FID) analysis of ArJ essential oil revealed the major classes of compounds as oxygenated monoterpenes (>57%) and cinnamic acid derivatives (18.03%). The antimicrobial tests of ArJ essential oil revealed that
,
, and
were the most susceptible test organisms. Two second-degree burns (each 1 inch square in diameter) were created on the dorsum of rats using an aluminum cylinder heated to 120 °C for 10 s. The wounds were treated either with ArJ or SS ointments for 21 days, while the negative control remained untreated, and biopsies were obtained for histological and biochemical analysis. The ArJ group demonstrated a significant increase in antioxidant superoxide dismutase (SOD) and catalase (CAT) enzymatic activities, while lipid peroxide (LP) levels remained insignificant compared to the negative control group. Additionally, ArJ and SS groups demonstrated a significant decrease in inflammatory levels of tumor necrosis factor α (TNF-α) compared to the negative group, while interleukin 1 beta (IL-1b) and IL-6 were comparable to the negative group. At the same time, anti-inflammatory IL-10 and transforming growth factor beta 1 (TGF-b1) markers increased significantly in the ArJ group compared to the negative control. The ArJ results demonstrated potent wound healing effects, comparable to SS, attributable to antioxidant and anti-inflammatory effects as well as a high proportion of oxygenated monoterpenes and cinnamate derivatives.
Abstract
1,25-Dihydroxyvitamin D3 1,25(OH)2D3, the active form of vitamin D, exerts potent effects on several tissues including cells of the immune system, where it affects T cell activation, ...differentiation and migration. The circulating, inactive form of vitamin D, 25(OH)D3, is generally used as an indication of vitamin D status. However, use of this precursor depends on its uptake by cells and subsequent conversion by the enzyme 25(OH)D3-1α-hydroxylase (CYP27B1) into active 1,25(OH)2D3. Using human T cells, we show in this study that addition of inactive 25(OH)D3 is sufficient to alter T cell responses only when dendritic cells (DCs) are present. Mechanistically, CYP27B1 is induced in DCs upon maturation with LPS or upon T cell contact, resulting in the generation and release of 1,25(OH)2D3, which subsequently affects T cell responses. In most tissues, vitamin D binding protein acts as a carrier to enhance the use of vitamin D. However, we show that vitamin D binding protein modulates T cell responses by restricting the availability of inactive 25(OH)D3 to DC. These data indicate that the level of free 25(OH)D3 available to DCs determines the inflammatory/regulatory balance of ensuing T cell responses.
The active form of vitamin D, 1,25-dihydroxyvitamin D(3) (1,25(OH)(2)D(3)), has potent immunomodulatory properties that have promoted its potential use in the prevention and treatment of infectious ...disease and autoimmune conditions. A variety of immune cells, including macrophages, dendritic cells, and activated T cells express the intracellular vitamin D receptor and are responsive to 1,25(OH)(2)D(3.) Despite this, how 1,25(OH)(2)D(3) regulates adaptive immunity remains unclear and may involve both direct and indirect effects on the proliferation and function of T cells. To further clarify this issue, we have assessed the effects of 1,25(OH)(2)D(3) on human CD4(+)CD25(-) T cells. We observed that stimulation of CD4(+)CD25(-) T cells in the presence of 1,25(OH)(2)D(3) inhibited production of proinflammatory cytokines including IFN- gamma, IL-17, and IL-21 but did not substantially affect T cell division. In contrast to its inhibitory effects on inflammatory cytokines, 1,25(OH)(2)D(3) stimulated expression of high levels of CTLA-4 as well as FoxP3, the latter requiring the presence of IL-2. T cells treated with 1,25(OH)(2)D(3) could suppress proliferation of normally responsive T cells, indicating that they possessed characteristics of adaptive regulatory T cells. Our results suggest that 1,25(OH)(2)D(3) and IL-2 have direct synergistic effects on activated T cells, acting as potent anti-inflammatory agents and physiologic inducers of adaptive regulatory T cells.
The cytolytic ionotropic ATP receptor P2X 7 has several important roles in immune cell regulation, such as cytokine release, apoptosis, and microbial killing. Although
P2X 7 receptors are frequently ...coexpressed with another subtype of P2X receptor, P2X 4 , they are believed not to form heteromeric assemblies but to function only as homomers. Both receptors play a role in neuropathic
pain; therefore, understanding how they coordinate the cellular response to ATP is important for the development of effective
pain therapies. Here, we provide biochemical and electrophysiological evidence for an association between P2X 4 and P2X 7 that increases the diversity of receptor currents mediated via these two subtypes. The heterologously expressed receptors
were coimmunoprecipitated from human embryonic kidney (HEK) 293 cells, and the endogenous P2X 4 and P2X 7 receptors were similarly coimmunoprecipitated from bone marrow-derived macrophages. In HEK293 cells, the fraction of P2X 4 receptors biotinylated at the plasma membrane increased 2-fold in the presence of P2X 7 although there was no change in overall expression. Coexpression of a dominant-negative P2X 4 mutant (C353W) with P2X 7 , inhibited P2X 7 receptor mediated currents by greater than 2-fold, whereas a nonfunctional but nonâdominant-negative mutant (S341W) did not.
Coexpression of P2X 4 S341W with P2X 7 produced a current that was potentiated by ivermectin and inhibited by 2â²,3â²- O -(2,4,6-trinitrophenyl) adenosine 5-triphosphate (TNP-ATP), whereas expression of P2X 7 alone produced a current that was insensitive to both of these compounds at the concentrations used. These results demonstrate
a structural and functional interaction between P2X 4 and P2X 7 , which suggests that they associate to form heteromeric receptors.
CTLA‐4 is an essential regulator of T‐cell immune responses whose intracellular trafficking is a hallmark of its expression. Defects in CTLA‐4 trafficking due to LRBA deficiency cause profound ...autoimmunity in humans. CTLA‐4 rapidly internalizes via a clathrin‐dependent pathway followed by poorly characterized recycling and degradation fates. Here, we explore the impact of manipulating Rab GTPases and LRBA on CTLA‐4 expression to determine how these proteins affect CTLA‐4 trafficking. We observe that CTLA‐4 is distributed across several compartments marked by Rab5, Rab7 and Rab11 in both HeLa and Jurkat cells. Dominant negative (DN) inhibition of Rab5 resulted in increased surface CTLA‐4 expression and reduced internalization and degradation. We also observed that constitutively active (CA) Rab11 increased, whereas DN Rab11 decreased CTLA‐4 surface expression via an impact on CTLA‐4 recycling, indicating CTLA‐4 shares similarities with other recycling receptors such as EGFR. Additionally, we studied the impact of manipulating both LRBA and Rab11 on CTLA‐4 trafficking. In Jurkat cells, LRBA deficiency was associated with markedly impaired CTLA‐4 recycling and increased degradation that could not be corrected by expressing CA Rab11. Moreover LRBA deficiency reduced CTLA‐4 colocalization with Rab11, suggesting that LRBA is upstream of Rab11. These results show that LRBA is required for effective CTLA‐4 recycling by delivering CTLA‐4 to Rab11 recycling compartments, and in its absence, CTLA‐4 fails to recycle and undergoes degradation.
Correct trafficking of CTLA‐4 is required for its function and mutations in proteins such as LRBA lead to CTLA‐4 deficiency disorders. Here we detail the intracellular pathways involved in CTLA‐4 trafficking using constitutively active and dominant negative Rab GTPases to probe the relationship between CTLA‐4 and LRBA. Our data show CTLA‐4 recycling is strongly dependent on Rab11 activity and that LRBA function is likely upstream of Rab11.
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BFBNIB, DOBA, FZAB, GIS, IJS, IZUM, KILJ, NLZOH, NUK, OILJ, PILJ, PNG, SAZU, SBCE, SBMB, SIK, UILJ, UKNU, UL, UM, UPUK
Regulatory T cells (Treg) have a central role in controlling the activation of self-reactive T cells and maintaining peripheral tolerance in our body. Many effector mechanisms for Treg function have ...been described including a role for the protein CTLA-4 which is constitutively expressed by these cells. Despite its importance, there is currently little consensus in the methods and protocols for studying CTLA-4 function, which is partially due to debate over CTLA-4 function itself. In this chapter, we outline protocols used in our lab to study CTLA-4 function, which have been generated based on the observation that CTLA-4 acts to physically remove and degrade its ligands expressed by antigen presenting cells. Accordingly, we provide protocols for isolation of human monocytes and their differentiation into dendritic cells (DC), purification of conventional and regulatory T-cell populations, and the assembly of CTLA-4-dependent Treg suppression assays. We hope that this will offer a reliable platform for dissecting the biology of CTLA-4 on Treg and for testing reagents aimed at modulating CTLA-4 function. Such assays are increasingly vital for the study of immune function in both healthy individuals and patients with a variety of autoimmune and immune dysregulation syndromes.
Manipulation of the CD28/CTLA-4 pathway is at the heart of a number of immunomodulatory approaches used in both autoimmunity and cancer. Although it is clear that CTLA-4 is a critical regulator of T ...cell responses, the immunological contexts in which CTLA-4 controls immune responses are not well defined. In this study, we show that whereas CD80/CD86-dependent activation of resting human T cells caused extensive T cell proliferation and robust CTLA-4 expression, in this context CTLA-4 blocking Abs had no impact on the response. In contrast, in settings where CTLA-4(+) cells were present as "regulators," inhibition of resting T cell responses was dependent on CTLA-4 expression and specifically related to the number of APC. At low numbers of APC or low levels of ligand, CTLA-4-dependent suppression was highly effective whereas at higher APC numbers or high levels of ligand, inhibition was lost. Accordingly, the degree of suppression correlated with the level of CD86 expression remaining on the APC. These data reveal clear rules for the inhibitory function of CTLA-4 on regulatory T cells, which are predicted by its ability to remove ligands from APC.
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