Identification and functional characterization of CD8+ T regulatory cells in type 1 diabetes patients.

Type 1 diabetes is an autoimmune disease where autoreactive T lymphocytes destroy pancreatic beta cells. We previously reported a defect in CD4+ Tregs cell proliferation and reduced CD4+ Tregs PD-1 expression in patients. Another 'memory-like' regulatory subset, CD8+ Tregs, evaluated as CD8+CD25+FOXP3+, has recently raised interest for their effective suppressive activity. Different CD8+ T cell populations, their proliferation capacity and expression of PD-1 molecule were evaluated by flow-cytometer analysis in newly diagnosed, long-term Type 1 diabetes patients compared to healthy normal donors. Under basal conditions, CD8+ Tregs and CD8+ Teffs were seemingly represented among study groups while there was evidence of diminished expression of PD-1 in Teff subsets of long-term patients. After 3 days of PMA/ionomycin stimulation, patients CD8+ Tregs showed decreased percentage in respect to control group. CD8+ Teffs were instead increased in long-term diabetics versus controls. PD-1+CD8+ Tregs were represented at a much lower percentage in long-term diabetic patients, in respect to controls. Importantly, patients CD8+ Tregs and CD8+ Teffs presented a significant proliferation defect in respect to the control group. In conclusion, our study indicates that a defect of CD8+ Tregs is observed in diabetics. This subset could thus represent a novel target of immunotherapy in patients.

Reversion of resistance to immunosuppressive agents in three patients with psoriatic arthritis by cyclosporine A: modulation of P-glycoprotein function.

Secondary resistance may be a major problem in the management of autoimmune diseases. P-glycoprotein (P-gp) over-function has been described as a mechanism of drug resistance in autoimmune patients. P-gp function can in vitro be inhibited by cyclosporine A (CSA) and verapamil; moreover, P-gp reduction by CSA in systemic lupus erythematosus and rheumatoid arthritis has been demonstrated. Here, P-gp function before and after CSA administration in three psoriatic arthritis (PsA) patients, who developed a resistance to MTX/SSA, has been evaluated. P-gp function on patient cells was analyzed by measuring the changes in rhodamine-123 (Rh-123) fluorescence after verapamil incubation. CSA treatment resulted in good clinical outcome that was related with a significant P-gp function reduction at CD3+ and CD8+ levels. In addition to its immunosuppressive activity, CSA results may also be related to MTX/SSA effect restoration through P-gp inhibition. This is the first time that CSA has been demonstrated as being able to revert MTX/SSA resistance in PsA.

Increased expression of mucosal addressin cell adhesion molecule 1 in the duodenum of patients with active celiac disease is associated with depletion of integrin alpha4beta7-positive T cells in blood.

Mucosal addressin cell adhesion molecule 1, expressed on gut endothelial cells, in conjunction with integrin alpha(4)beta(7), expressed on lymphocytes, is critical in lymphocyte homing to the gut. The mucosal addressin cell adhesion molecule 1/integrin alpha(4)beta(7) pathway is involved in the pathogenesis of chronic intestinal inflammation by recruiting lymphocytes into inflamed gut. We explored the duodenal expression of mucosal addressin cell adhesion molecule 1 and the peripheral T-cell expression of integrin alpha(4)beta(7) in patients with celiac disease. Duodenal biopsies and a peripheral blood sample were collected from 15 celiac patients, before and after 12 months of gluten-free diet, and from 12 control subjects. Treated celiac biopsies were cultured with peptic-tryptic digest of gliadin and/or an anti-interferon alpha neutralizing antibody. Mucosal addressin cell adhesion molecule 1 was determined by confocal immunofluorescence microscopy and immunoblotting. Integrin beta(7)-positive T cells were analyzed by flow cytometry. Mucosal addressin cell adhesion molecule 1 expression was significantly higher in active celiac disease than in normal mucosa. After gluten-free diet, a dramatic reduction of mucosal addressin cell adhesion molecule 1 was also observed. No difference was seen between patients with celiac disease after treatment and controls. Ex vivo peptic-tryptic digest of gliadin challenge induced a marked increase of mucosal addressin cell adhesion molecule 1 expression. Blocking interferon alpha inhibited the peptic-tryptic digest of gliadin-induced mucosal addressin cell adhesion molecule 1 overexpression. The percentage of circulating beta(7)-positive T cells was significantly lower in untreated celiac disease in comparison to controls but normalized after gluten-free diet. Mucosal addressin cell adhesion molecule 1 is strongly up-regulated in active celiac disease dependent on interferon alpha and is associated with peripheral depletion of integrin alpha(4)beta(7)-expressing T cells. We conclude that mucosal addressin cell adhesion molecule 1 may represent an important determinant for the generation of mucosal damage in celiac disease.

CXCL13, CCL21, and CXCL12 expression in salivary glands of patients with Sjogren's syndrome and MALT lymphoma: association with reactive and malignant areas of lymphoid organization.

The chemokines (CKs) CXCL13, CCL21, and CXCL12 are known to play differential roles in the organization of the lymphoid tissues and the development of lymphoid malignancies. We investigated the expression of these CKs and their receptors in the salivary glands of Sjogren's syndrome patients with lymphoepithelial lesions (lymphoepithelial sialadenitis or LESA) and in MALT lymphoma to understand their involvement in salivary gland lymphomagenesis. We demonstrate that within salivary glands with LESA and MALT lymphoma the lymphoid CKs CXCL13 and CCL21 are selectively associated with areas of reactive lymphoid proliferation, whereas no significant expression of these molecules was detected in the malignant lymphoid aggregate. Conversely, CXCL12 was observed predominantly in infiltrated ducts and malignant B cells. Accordingly, CXCL13 and CCL21 transcript levels were significantly increased in LESA samples while CXCL12 levels were increased in MALT lymphoma and isolated tumor cells. Low levels of CK receptors were detected on lymphoma-extracted lymphocytes, suggesting down-regulation in the abundance of ligands. Our findings suggest that in salivary gland MALT lymphoma the lymphoid CKs CXCL13 and CCL21 are directly implicated in the organization of ectopic reactive lymphoid tissue, whereas CXCL12 is associated with the infiltrated epithelium and malignant B cell component and is possibly involved in the regulation of malignant B cell survival.

B-cell homeostasis, competition, resources, and positive selection by self-antigens.

In adult mice, the number of B lymphocytes remains constant under homeostatic control, in spite of the fact that B cells are produced continuously in numbers that largely exceed the number required to replenish the peripheral pools. It follows that each newly formed lymphocyte can only persist if another lymphocyte dies. In an immune system where the total number of cells is limited, cell survival is no longer a passive phenomenon but rather a continuous active process where each lymphocyte must compete with other lymphocytes to survive. Consequently, the number and the life expectancy of a B-cell clone vary according to the presence or absence of competitor populations. This process of lymphocyte competition is likely controlled by a common need for resources that are in limited supply. The number of peripheral B-cells varies according to the availability of B-cell receptor (BCR) ligands. Indeed, it is possible to modify steady-state B-cell numbers by antigen manipulation. Moreover, conventional self-reactive B cells can undergo positive selection. We showed that the fate of a self-reactive B cell is determined by the quantity of self-antigens, the number of antigen-specific receptors engaged, and its overall antigen-binding avidity rather than the affinity of individual BCRs.