Purinergic Profiling of Regulatory T-cells in Patients With Episodic Migraine.
ABSTRACT: Objectives: Immune responses in migraine are poorly characterized, yet implicated in the disease pathogenesis. This study was carried out to characterize purinergic profiles of T-cells in patients with episodic migraine without aura (MWoA) to provide mechanistic evidence for ATP and adenosine involvement in modulation of immune regulation in migraine. Methods: Peripheral blood samples were obtained from patients with migraine (n = 16) and age-matched control subjects (n = 21). Subsets of T-cells were identified by flow cytometry based on specific membrane markers. Results: Migraine patients showed reduced total T-cell counts in the peripheral blood. Whereas the total number of CD3+CD4+, CD3+CD8+, or regulatory T lymphocytes (Treg) was not changed, the proportion of Treg CD45R0+CD62L- and CD45R0-CD62L- cells was increased. Interestingly, in migraine, less Treg cells expressed CD39 and CD73 suggesting disrupted ATP breakdown to adenosine. The negative correlations were observed between the duration of migraine and the relative number of CD73+CD39- Tregs and total number of CD73-positive CD45R0+CD62L+ Tregs. Conclusion: Obtained data indicate that T-cell populations are altered in episodic migraine and suggest the involvement of Tregs in the pathophysiology of this disorder. Reduced expression of CD39 and CD73 suggests promotion of ATP-dependent pro-inflammatory and reduction of adenosine-mediated anti-inflammatory mechanisms in migraine.
Project description:Foxp3(+) regulatory T cells (Tregs) express both ectoenzymes CD39 and CD73, which in tandem hydrolyze pericellular ATP into adenosine, an immunoinhibitory molecule that contributes to Treg suppressive function. Using Foxp3GFP knockin mice, we noted that the mouse CD4(+)CD39(+) T-cell pool contains two roughly equal size Foxp3(+) and Foxp3(-) populations. While Foxp3(+)CD39(+) cells are CD73(bright) and are the bone fide Tregs, Foxp3(-)CD39(+) cells do not have suppressive activity and are CD44(+)CD62L(-)CD25(-)CD73(dim/-), exhibiting memory cell phenotype. Functionally, CD39 expression on memory and Treg cells confers protection against ATP-induced apoptosis. Compared with Foxp3(-)CD39(-) naïve T cells, Foxp3(-)CD39(+) cells freshly isolated from non-immunized mice express at rest significantly higher levels of mRNA for T-helper lineage-specific cytokines IFN-gamma (Th1), IL-4/IL-10 (Th2), IL-17A/F (Th17), as well as pro-inflammatory cytokines, and rapidly secrete these cytokines upon stimulation. Moreover, the presence of Foxp3(-)CD39(+) cells inhibits TGF-beta induction of Foxp3 in Foxp3(-)CD39(-) cells. Furthermore, when transferred in vivo, Foxp3(-)CD39(+) cells rejected MHC-mismatched skin allografts in a much faster tempo than Foxp3(-)CD39(-) cells. Thus, besides Tregs, CD39 is also expressed on pre-existing memory T cells of Th1-, Th2- and Th17-types with heightened alloreactivity.
Project description:The study aimed to compare composition of peripheral blood T-cell subsets and assess their surface expression of CD39 and CD73 ectonucleotidases in patients with severe and moderate aortic stenosis (AS) as well as to evaluate involvement of T-cell-mediated immune processes in valve calcification. The study was performed with 38 patients suffering from severe calcified aortic stenosis (SAS), 33 patients with MAS, and 30 apparently healthy volunteers (HVs). The relative distribution and percentage of T-cell subsets expressing CD39 and CD73 were evaluated by flow cytometry. T helper (Th) and cytotoxic T-cell subsets (Tcyt) were identified by using CD3, CD4, and CD8 antibodies. Regulatory T cells (Tregs) were characterized by the expression of CD3, CD4, and high IL-2R alpha chain (CD25high) levels. CD45R0 and CD62L were used to assess differentiation stage of Th, Tcyt, and Treg subsets. It was found that MAS and SAS patients differed in terms of relative distribution of Tcyt and absolute number of Treg. Moreover, the absolute number of Tcyt and terminally differentiated CD45RA-positive effector T-cells (TEMRA) subset was significantly higher in SAS vs. MAS patients and HVs. However, the absolute and relative number of naïve Th and the absolute number of Treg were significantly higher in MAS vs. SAS patients; the relative number of naïve Tregs was significantly (<i>p</i> < 0.01) decreased in SAS patients. It was shown that CD73 expression was significantly higher in SAS vs. MAS patients noted in all EM, CM, TEMRA, and naïve Th cell subsets. However, only the latter were significantly increased (<i>p</i> = 0.003) in patients compared with HVs. SAS vs. MAS patients were noted to have significantly higher percentage of CD73+ EM Tcyt (<i>p</i> = 0.006) and CD73+ CM Tcyt (<i>p</i> = 0.002). The expression of CD73 in patients significantly differed in all three Treg populations such as EM (<i>p</i> = 0.049), CM (<i>p</i> = 0.044), and naïve (<i>p</i> < 0.001). No significant differences in CD39 expression level was found in MAS and SAS patients compared with the HV group. Overall, the data obtained demonstrated that purinergic signaling was involved in the pathogenesis of aortic stenosis and calcification potentially acting <i>via</i> various cell types, wherein among enzymes, degrading extracellular ATP CD73 rather than CD39 played a prominent role.
Project description:Adenosine acts as anti-inflammatory mediator on the immune system and has been described in regulatory T cell (Treg)-mediated suppression. In the absence of adenosine deaminase (ADA), adenosine and other purine metabolites accumulate, leading to severe immunodeficiency with recurrent infections (ADA-SCID). Particularly ADA-deficient patients with late-onset forms and after enzyme replacement therapy (PEG-ADA) are known to manifest immune dysregulation. Herein we provide evidence that alterations in the purine metabolism interfere with Treg function, thereby contributing to autoimmune manifestations in ADA deficiency. Tregs isolated from PEG-ADA-treated patients are reduced in number and show decreased suppressive activity, whereas they are corrected after gene therapy. Untreated murine ADA(-/-) Tregs show alterations in the plasma membrane CD39/CD73 ectonucleotidase machinery and limited suppressive activity via extracellular adenosine. PEG-ADA-treated mice developed multiple autoantibodies and hypothyroidism in contrast to mice treated with bone marrow transplantation or gene therapy. Tregs isolated from PEG-ADA-treated mice lacked suppressive activity, suggesting that this treatment interferes with Treg functionality. The alterations in the CD39/CD73 adenosinergic machinery and loss of function in ADA-deficient Tregs provide new insights into a predisposition to autoimmunity and the underlying mechanisms causing defective peripheral tolerance in ADA-SCID.
Project description:A previous report has shown that regulatory T cells (Treg) were markedly more sensitive to adenosine-5'-triphosphate (ATP) than conventional T cells (Tconv). Another one has shown that Tregs and CD45RBlow Tconvs, but not CD45RBhigh Tconvs, displayed similar high sensitivity to ATP. We have previously reported that CD45RBlow Tconvs expressing B220/CD45RABC molecules in a pre-apoptotic stage are resistant to ATP stimulation due to the loss of P2X7 receptor (P2X7R) membrane expression. To gain a clearer picture on T-cell sensitivity to ATP, we have quantified four different cellular activities triggered by ATP in mouse T cells at different stages of activation/differentiation, in correlation with levels of P2X7R membrane expression. P2X7R expression significantly increases on Tconvs during differentiation from naive CD45RBhighCD44low to effector/memory CD45RBlowCD44high stage. Maximum levels of upregulation are reached on recently activated CD69+ naive and memory Tconvs. Ectonucleotidases CD39 and CD73 expression levels increase in parallel with those of P2X7R. Recently activated CD69+ CD45RBhighCD44low Tconvs, although expressing high levels of P2X7R, fail to cleave homing receptor CD62L after ATP treatment, but efficiently form pores and externalize phosphatidylserine (PS). In contrast, naive CD45RBhighCD44low Tconvs cleave CD62L with high efficiency although they express a lower level of P2X7, thus suggesting that P2X7R levels are not a limiting factor for signaling ATP-induced cellular responses. Contrary to common assumption, P2X7R-mediated cellular activities in mouse Tconvs are not triggered in an all-or-none manner, but depend on their stage of activation/differentiation. Compared to CD45RBlow Tconvs, CD45RBlowFoxp3+ Tregs show significantly higher levels of P2X7R membrane expression and of sensitivity to ATP as evidenced by their high levels of CD62L shedding, pore formation and PS externalization observed after ATP treatment. In summary, the different abilities of ATP-treated Tconvs to form pore or cleave CD62L depending on their activation and differentiation state suggests that P2X7R signaling varies according to the physiological role of T convs during antigen activation in secondary lymphoid organs or trafficking to inflammatory sites.
Project description:Injection of regulatory T cells (Tregs) followed by sensitization with 2,4,6-trinitrochlorobenzene induced a transient increase in size and cellularity of skin-draining lymph nodes (LNs) in mice. This led us to hypothesize that Tregs may affect the trafficking of T cells from and to peripheral LNs. Two to three hours after sensitization, we found fewer CD8+ T cells expressing CD62L in LNs compared with untreated controls. Injection of wild-type Tregs prevented this down-regulation of CD62L. In contrast, Tregs devoid of the adenosine triphosphate (ATP)-degrading ecto-enzyme CD39 were unable to do so. As for the mechanism of CD62L regulation, we found that ATP, which is released in skin upon hapten-exposure, is inducing the protease ADAM17 in LN-residing T cells via engagement of P2X7 ATP receptors. ADAM17 cleaves CD62L from the surface of CD8+ T cells, which in turn provide a signal for T cells to leave the LNs. This regulation of CD62L is disturbed by the presence of Tregs, because Tregs remove extracellular ATP from the tissue by activity of CD39 and, therefore, abrogate the shedding of CD62L. Thus, these data indicate that the regulation of ATP turnover by Tregs in skin and LNs is an important modulator for immune responses.
Project description:PURPOSE:Regulatory T cell (Treg) frequency and activity are increased in cancer patients and play a major role in tumor escape. Although disease progression is favored by the presence of Treg, mechanisms used by Treg to suppress antitumor immunity are unknown. The ectonucleotidases CD39 and CD73 are expressed in Treg and convert ATP into immunosuppressive adenosine. In this study, the involvement of the adenosinergic pathway in Treg-mediated suppression in head and neck squamous cell carcinoma (HNSCC) patients was evaluated. EXPERIMENTAL DESIGN:HNSCC patients with an active disease (n = 19) and patients with no evident disease after therapy (n = 14) were studied. Ectonucleotidase expression on CD4(+) T cells and CD4(+)CD25(high) Treg was evaluated by flow cytometry and compared with normal controls. Ectonucleotidase activity was also compared within these three groups. The data were analyzed for associations of ectonucleotidase expression/function with disease stage. RESULTS:The percentages and expression levels of CD39 and CD73 in CD4(+) T cells and Treg were greater in HNSCC than in normal controls and highest in patients with no evident disease. Patients' Treg hydrolyzed ATP at higher rates and produced higher levels of adenosine than normal controls' Treg. The increased frequency and enzymatic activity of CD4(+)CD39(+) cells corresponded to increased adenosine-mediated suppression of effector T cells, which was partly inhibited by ARL67156, an ectonucleotidase inhibitor, and by ZM241385, a selective A(2a)/A(2b) receptor antagonist. CONCLUSIONS:CD39(+) Treg frequency and adenosine-mediated suppression are significantly increased in HNSCC patients. The adenosinergic pathway is involved in Treg-mediated immunosuppression in cancer and its attenuation could be a promising immunotherapeutic strategy for patients with HNSCC.
Project description:Adult T-cell leukemia/lymphoma (ATLL) patients have an extremely poor prognosis, partly due to their immunosuppressive state. The majority of ATLL patients have leukemic cells with phenotype similar to Tregs, prompting suggestions that ATLL cells themselves have immunosuppressive functions. In this study, we detected CD39 expression on ATLL cells, particularly frequent on aggressive subtypes. CD39 and CD73 convert extracellular adenosine triphosphate (ATP) into adenosine, a key player in Tregs' immunosuppression. In vitro culture, both CD39<sup>+</sup> ATLL cells and normal Tregs converted rapidly extracellular ATP to AMP, which was disturbed by CD39 inhibitors, and was negated in the CD39 knockout MJ cell line. The proliferation of cocultured CD4<sup>+</sup>/CD8<sup>+</sup> normal T cells was suppressed by CD39<sup>+</sup> MJ cells, but not by CD39 knockout MJ cells. Supplemented ATP was exhausted by an EG7-OVA T-cell line with stable CD39 induction, but not by mock. When these cell lines were subcutaneously transplanted into murine flanks, Poly(I:C) peritoneal administration reduced tumor size to 1/3 in mock-transplanted tumors, but not in CD39 induced tumors. Overall, we found that ATLL cells express CD39 at a high rate, and our results suggest that this helps ATLL cells escape antitumor immunity through the extracellular ATPDase-Adenosine cascade. These findings will guide future clinical strategies for ATLL treatment.
Project description:The mechanisms by which Regulatory T cells suppress IL-2 production of effector CD4+ T cells in pathological conditions are unclear. A subpopulation of human Treg expresses the ectoenzyme CD39, which in association with CD73 converts ATP/ADP/AMP to adenosine. We show here that Treg/CD39+ suppress IL-2 expression of activated CD4+ T-cells more efficiently than Treg/CD39-. This inhibition is due to the demethylation of an essential CpG site of the il-2 gene promoter, which was reversed by an anti-CD39 mAb. By recapitulating the events downstream CD39/adenosine receptor (A2AR) axis, we show that A2AR agonist and soluble cAMP inhibit CpG site demethylation of the il-2 gene promoter. A high frequency of Treg/CD39+ is associated with a low clinical outcome in HIV infection. We show here that CD4+ T-cells from HIV-1 infected individuals express high levels of A2AR and intracellular cAMP. Following in vitro stimulation, these cells exhibit a lower degree of demethylation of il-2 gene promoter associated with a lower expression of IL-2, compared to healthy individuals. These results extend previous data on the role of Treg in HIV infection by filling the gap between expansion of Treg/CD39+ in HIV infection and the suppression of CD4+ T-cell function through inhibition of IL-2 production.
Project description:In the tumor microenvironment (TME) ATP and its receptor P2X7 exert a pivotal influence on cancer growth and tumor-host interactions. Here we analyzed the different effect of P2X7 genetic deficiency versus its antagonism on response against P2X7-expressing implanted tumors. We focused on immune cell expression of ATP degrading enzymes CD39 and CD73 and in vivo measured TME's ATP. The immune infiltrate of tumors growing in P2X7 null mice shows a decrease in CD8+ cells and an increased number of Tregs, overexpressing the fitness markers OX40, PD-1, and CD73. A similar Treg phenotype is also present in the spleen of tumor-bearing P2X7 null mice and it is paralleled by a decrease in proinflammatory cytokines and an increase in TGF-?. Differently, systemic administration of the P2X7 blocker A740003 in wild-type mice left unaltered the number of tumor-infiltrating CD8+ and Treg lymphocytes but increased CD4+ effector cells and decreased their expression of CD39 and CD73. P2X7 blockade did not affect spleen immune cell composition or ectonucleotidase expression but increased circulating INF-?. Augmented CD73 in P2X7 null mice was mirrored by a decrease in TME ATP concentration and nucleotide reduced secretion from immune cells. On the contrary, TME ATP levels remained unaltered upon P2X7 antagonism, owing to release of ATP from cancerous cells and diminished ectonucleotidase expression by CD4+ and dendritic cells. These data point at P2X7 receptor as a key determinant of TME composition due to its combined action on immune cell infiltrate, ectonucleotidases, and ATP release.
Project description:Microglial cells invade the brain as amoeboid precursors and acquire a highly ramified morphology in the postnatal brain. Microglia express all essential purinergic elements such as receptors, nucleoside transporters and ecto-enzymes, including CD39 (NTPDase1) and CD73 (5'-nucleotidase), which sequentially degrade extracellular ATP to adenosine. Here, we show that constitutive deletion of CD39 and CD73 or both caused an inhibition of the microglia ramified phenotype in the brain with a reduction in the length of processes, branching frequency and number of intersections with Sholl spheres. In vitro, unlike wild-type microglia, cd39-/- and cd73-/- microglial cells were less complex and did not respond to ATP with the transformation into a more ramified phenotype. In acute brain slices, wild-type microglia retracted approximately 50% of their processes within 15 min after slicing of the brain, and this phenomenon was augmented in cd39-/- mice; moreover, the elongation of microglial processes towards the source of ATP or towards a laser lesion was observed only in wild-type but not in cd39-/- microglia. An elevation of extracellular adenosine 1) by the inhibition of adenosine transport with dipyridamole, 2) by application of exogenous adenosine or 3) by degradation of endogenous ATP/ADP with apyrase enhanced spontaneous and ATP-induced ramification of cd39-/- microglia in acute brain slices and facilitated the transformation of cd39-/- and cd73-/- microglia into a ramified process-bearing phenotype in vitro. These data indicate that under normal physiological conditions, CD39 and CD73 nucleotidases together with equilibrative nucleoside transporter 1 (ENT1) control the fate of extracellular adenosine and thereby the ramification of microglial processes.