Quantitative and functional analysis of CD69(+) NKG2D(+) T regulatory cells in healthy subjects.

T regulatory (Treg) cells have a key role in immune homeostasis and the pathogenesis of chronic inflammatory and autoimmune diseases. CD69 is an early leukocyte activation molecule that under steady state conditions is detected in a small proportion of lymphocytes in peripheral blood and lymphoid tissues. Although it has been reported that a subset of CD69(+) T cells behaves as Treg lymphocytes, the possible relationship between CD69(+) Treg cells and CD4(+)NKG2D(+) T lymphocytes, which also exert immunosuppressive activity, has not been explored. In this study, we analyzed the expression of CD69 and NKG2D by T lymphocytes from the peripheral blood of twenty-five healthy subjects by multi-parametric flow cytometry analysis, and their suppressive activity by an assay of inhibition of lymphocyte activation (CD40L expression) and proliferation (carboxyfluorescein partition assay). We found a very small percentage of CD4(+)CD69(+)NKG2D(+) T cells (median 0.002%, Q1-Q3, 0.001-0.004%), which also expressed TGF-ß (Latency Associated Peptide or LAP) and IL-10, in all samples analyzed. These cells exerted an important in vitro suppressive effect on both activation and proliferation of T effector cells. Our data suggest that at very small numbers, CD4(+)CD69(+)NKG2D(+) lymphocytes seem to exert a relevant functional immune-regulatory role in healthy subjects.

Analysis of expression and function of the co-stimulatory receptor SLAMF1 in immune cells from patients with systemic lupus erythematosus (SLE).

The signaling lymphocytic activation molecule SLAMF1 (CD150) is a co-stimulatory molecule that is expressed by most immune cells, including T regulatory (Treg) lymphocytes. Since different abnormalities have been reported regarding the number and function of Foxp3+ Treg cells in patients with systemic lupus erythematosus (SLE), we decided to analyze the expression and function of CD150 in these regulatory lymphocytes in this condition. We isolated peripheral blood mononuclear cells from 20 patients with SLE, and 20 healthy controls. The expression of SLAMF1 was determined by multi-parametric flow cytometry and the suppressive function of CD4+CD25+ lymphocytes, upon engagement or not of CD150 with an agonistic monoclonal antibody, was analyzed by an assay of inhibition of cell proliferation. We observed a significantly increased expression of SLAMF1 by CD3+CD4+ helper T cells and CD19+ B cells in patients with SLE and active disease. However, similar levels of SLAMF1 expression were detected in Foxp3+ Treg cells from patients and controls. In contrast, a higher proportion of SLE patients increased their suppressive function of Treg cells upon CD150 engagement compared to healthy controls. Our data suggest that SLAMF1 is another significant piece in the intricate defective immune-regulatory function of patients with SLE.

Proportions of several types of plasma and urine microparticles are increased in patients with rheumatoid arthritis with active disease.

We analysed the proportions of different microparticles (MPs) in plasma from patients with rheumatoid arthritis (RA), and assessed their relationship with disease activity/therapy and their in-vitro effect on proinflammatory cytokine release. Blood and urine samples were obtained from 55 patients with RA (24 untreated and 31 under conventional therapy) and 20 healthy subjects. Fourteen patients with systemic lupus erythematosus (SLE) were also studied. The proportions of CD3(+) , CD14(+) , CD19(+) , CD41(+) and CD62E(+) MPs were determined by flow cytometry analysis. The in-vitro effect of plasma MPs on the release of interleukin (IL)-1, IL-6, IL-17 and tumour necrosis factor (TNF)-α was also analysed. We detected that the proportions of different types of annexin-V(+) MPs were enhanced in plasma (CD3(+) , CD14(+) , CD19(+) , CD41(+) and CD62E(+) MPs) and urine (CD14(+) , CD3(+) and CD19(+) MPs) from RA patients with high disease activity (DAS28 index > 5·1). Accordingly, a significant positive correlation was observed between the levels of MPs and DAS28 score, and these levels diminished significantly at week 4 of immunosuppressive therapy. Finally, MPs isolated from patients with high disease activity induced, in vitro, an enhanced release of IL-1, IL-17 and TNF-α. In SLE, enhanced levels of different types of plasma MPs were also detected, with a tight correlation with disease activity. Our data further support that MPs have a relevant role in the pathogenesis of RA and suggest that the analysis of the proportions of these microvesicles in plasma could be useful to monitor disease activity and therapy response in patients with RA.

Expression and function of Cbl-b in T cells from patients with systemic lupus erythematosus, and detection of the 2126 A/G Cblb gene polymorphism in the Mexican mestizo population.

Systemic lupus erythematosus (SLE) is characterized by abnormalities in the function of T and B lymphocytes and in the signaling pathways induced through their receptors. Cbl-b is an intracellular adaptor protein that plays a key role in the negative regulation of lymphocyte activity. We explored the expression and function of Cbl-b in T lymphocytes from SLE patients. In addition, the possible association of SLE and a single nucleotide polymorphism (SNP) of the Cblb gene was determined. We studied 150 SLE patients, 163 healthy individuals, and 14 patients with rheumatoid arthritis (RA). The expression of Cbl-b was analyzed in the peripheral blood mononuclear cells, and the negative regulatory function of Cbl-b was assessed by analyzing actin polymerization and the phosphorylation of JNK and c-Jun induced through CD3. Furthermore, the 2126(A/G) SNP of the Cblb gene was detected by real-time polymerase chain reaction. We found a significant small reduction in the expression of Cbl-b as well as increased levels of activation of c-Jun and actin polymerization in T lymphocytes from patients with SLE compared with healthy controls or RA patients. In addition, a significant association between the 2126(A/G) SNP and SLE was detected. Our data suggest that Cbl-b may contribute to the deregulated activation of T lymphocytes observed in SLE.

Effect of arsenic on regulatory T cells.

INTRODUCTION: Arsenic (As) affects the function and survival of lymphocytes, and some arsenic compounds exert a relevant antineoplastic effect. We have explored the effect of As on T regulatory cells. RESULTS AND DISCUSSION: In vitro experiments with peripheral blood mononuclear cells from healthy subjects showed that low concentrations of As tended to increase the number of natural T regulatory (nTreg) lymphocytes, whereas concentrations >5.0 muM had an opposite effect. Furthermore, rats exposed to As showed redistribution of nTreg cells, and As administration to rats with experimental allergic encephalomyelitis increased the levels of nTreg cells in spleen and diminished the severity of this condition. On the other hand, in 47 apparently healthy subjects chronically exposed to As, we found significant inverse correlation between urinary As levels and the number and function of nTreg lymphocytes. Although most of these individuals showed enhanced levels of apoptotic lymphocytes in peripheral blood, with a diminution of mitochondrial membrane potential, no significant correlation between these parameters and urinary As was detected. CONCLUSION: Our data indicate that As seems to have a relevant and complex effect on nTreg cells.